Interferon α but Not Interleukin 12 Activates STAT4 Signaling in Human Vascular Endothelial Cells
2004; Elsevier BV; Volume: 279; Issue: 25 Linguagem: Inglês
10.1074/jbc.m401517200
ISSN1083-351X
AutoresNicholas Torpey, Stephen E. Maher, Alfred L.M. Bothwell, Jordan S. Pober,
Tópico(s)Cell Adhesion Molecules Research
ResumoSTAT4 signaling, activated by either interleukin 12 (IL12) or interferon α (IFNα), promotes TH1 responses in CD4+ T cells. Vascular endothelial cells (EC) may also become polarized in response to various cytokines, favoring recruitment and activation of TH1 or TH2 effector cells. Here we have investigated the role of the STAT4 pathway in EC. Cultured human umbilical vein EC (HUVEC) express low levels of STAT4, which may be tyrosine-phosphorylated by treatment with IFNα but not IL12. This is because HUVEC lack both subunits of the IL12 receptor (IL12Rβ1 and IL12Rβ2), even following treatment with various cytokines. IL12 phosphorylation of STAT4 can be observed in HUVEC that have been transduced to express the IL12R. To identify STAT4-induced genes we pursued three approaches: analysis by DNA microarray and quantitative RT-PCR (Q-PCR) of the IL12 responses in IL12R-transduced EC; analysis by Q-PCR of IFNα responses in STAT4-overexpressing EC; and analysis of IFNα responses in U3A neuroblastoma cell lines that express either STAT1 or STAT4, but not both. In all three instances we observe STAT4-mediated induction of the chemokine monocyte chemoattractant protein 1 (MCP1) and suppressor of cytokine signaling 3 (SOCS3) mRNA, and we confirm the production of each protein in both IL12R-transduced EC and STAT4-transduced U3A cells. These observations reveal that there is a STAT4 response of EC, activated by IFNα but not IL12, and that it may modulate the pro-inflammatory behavior of EC. STAT4 signaling, activated by either interleukin 12 (IL12) or interferon α (IFNα), promotes TH1 responses in CD4+ T cells. Vascular endothelial cells (EC) may also become polarized in response to various cytokines, favoring recruitment and activation of TH1 or TH2 effector cells. Here we have investigated the role of the STAT4 pathway in EC. Cultured human umbilical vein EC (HUVEC) express low levels of STAT4, which may be tyrosine-phosphorylated by treatment with IFNα but not IL12. This is because HUVEC lack both subunits of the IL12 receptor (IL12Rβ1 and IL12Rβ2), even following treatment with various cytokines. IL12 phosphorylation of STAT4 can be observed in HUVEC that have been transduced to express the IL12R. To identify STAT4-induced genes we pursued three approaches: analysis by DNA microarray and quantitative RT-PCR (Q-PCR) of the IL12 responses in IL12R-transduced EC; analysis by Q-PCR of IFNα responses in STAT4-overexpressing EC; and analysis of IFNα responses in U3A neuroblastoma cell lines that express either STAT1 or STAT4, but not both. In all three instances we observe STAT4-mediated induction of the chemokine monocyte chemoattractant protein 1 (MCP1) and suppressor of cytokine signaling 3 (SOCS3) mRNA, and we confirm the production of each protein in both IL12R-transduced EC and STAT4-transduced U3A cells. These observations reveal that there is a STAT4 response of EC, activated by IFNα but not IL12, and that it may modulate the pro-inflammatory behavior of EC. Vascular endothelial cells (EC) 1The abbreviations used are: EC, endothelial cells; LPS, lipopolysaccharide; STAT, signal transducer and activator of transcription; VSMC, vascular smooth muscle cells; HUVEC, human umbilical vein EC; HDMEC, human dermal microvascular endothelial cells; FCS, fetal calf serum; PBMC, peripheral blood mononuclear cells; SOCS, suppressor of cytokine signaling; RT-PCR, reverse transcriptase-PCR; MCP, monocyte chemoattractant protein; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein; IFN, interferon; PHA, phytohemagglutinin. are central to the development of inflammatory processes. In the resting state EC do not normally interact with circulating leukocytes. However, in response to inflammatory stimuli EC express cell surface adhesion molecules including E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) that allow for tethering and firm attachment of leukocytes, and ultimately their transmigration into the underlying tissues. The process of leukocyte recruitment by EC has been extensively reviewed (1Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6414) Google Scholar, 2Luscinskas F.W. Ma S. Nusrat A. Parkos C.A. Shaw S.K. Semin. Immunol. 2002; 14: 105-113Crossref PubMed Scopus (96) Google Scholar, 3Pober J.S. Immunol. Res. 1999; 19: 225-232Crossref PubMed Scopus (93) Google Scholar). In general, the inflammatory response promoted by cytokines such as tumor necrosis factor (TNF), or bacterial products, such as lipopolysaccharide (LPS), predominantly involves recruitment of neutrophils. However, the pro-inflammatory functions of human EC can be modulated by cytokines such as IFNγ, a TH1 cytokine, and IL4 and IL13, both TH2 cytokines, to favor recruitment of other cell types. Treatment of cultured EC with IFNγ enhances expression of E-selectin and ICAM-1 (4Doukas J. Pober J.S. J. Immunol. 1990; 145: 1727-1733PubMed Google Scholar), and the production of chemokines MIG, IP-10, and I-TAC (5Raju R. Malloy A. Shah T. Smith R. Oaks M. Hosenpud J.D. Transplantation. 2003; 75: 1072-1074Crossref PubMed Scopus (10) Google Scholar, 6Monaco C. Andreakos E. Young S. Feldmann M. Paleolog E. J. Leukoc. Biol. 2002; 71: 659-668PubMed Google Scholar), all of which may favor the recruitment of macrophages and possibly TH1 CD4+ lymphocytes. In contrast, IL4 treatment of EC attenuates E-selectin (7Bennett B.L. Cruz R. Lacson R.G. Manning A.M. J. Biol. 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Commun. 2002; 297: 1245-1252Crossref PubMed Scopus (55) Google Scholar), and DNA microarray studies in murine lymphocytes have identified more than 100 genes activated by IL12 whose expression is either directly or indirectly STAT4-dependent (21Hoey T. Zhang S. Schmidt N. Yu Q. Ramchandani S. Xu X. Naeger L.K. Sun Y.L. Kaplan M.H. EMBO J. 2003; 22: 4237-4248Crossref PubMed Scopus (78) Google Scholar). STAT4 can also be activated by type 1 interferons. At least in humans, STAT4 is recruited to the IFNα receptor (IFNAR) indirectly via receptor-associated STAT2, leading to STAT4 tyrosine phosphorylation and ultimately the activation of STAT4-dependent genes as above (22Farrar J.D. Smith J.D. Murphy T.L. Murphy K.M. J. Biol. Chem. 2000; 275: 2693-2697Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Farrar J.D. Smith J.D. Murphy T.L. Leung S. Stark G.R. Murphy K.M. Nat. Immunol. 2000; 1: 65-69Crossref PubMed Scopus (154) Google Scholar). The STAT4 signaling pathway plays a role in the polarization of CD4+ cell responses to an IFNγ-secreting TH1 pheno-type, and STAT4-deficient mice have impaired TH1 responses (24Kaplan M.H. Sun Y.L. Hoey T. Grusby M.J. Nature. 1996; 382: 174-177Crossref PubMed Scopus (1061) Google Scholar, 25Stamm L.M. Satoskar A.A. Ghosh S.K. David J.R. Satoskar A.R. Eur. J. Immunol. 1999; 29: 2524-2529Crossref PubMed Scopus (62) Google Scholar, 26Chitnis T. Najafian N. Benou C. Salama A.D. Grusby M.J. Sayegh M.H. Khoury S.J. J. Clin. Investig. 2001; 108: 739-747Crossref PubMed Scopus (188) Google Scholar). In addition to T lymphocytes, STAT4 is also expressed by B lymphocytes (27Durali D. de Goer de Herve M.G. Giron-Michel J. Azzarone B. Delfraissy J.F. Taoufik Y. Blood. 2003; 102: 4084-4089Crossref PubMed Scopus (80) Google Scholar), NK cells (16Cho S.S. Bacon C.M. Sudarshan C. Rees R.C. Finbloom D. Pine R. O'Shea J.J. J. Immunol. 1996; 157: 4781-4789PubMed Google Scholar), dendritic cells (DC), monocytes, and macrophages (28Frucht D.M. Aringer M. Galon J. Danning C. Brown M. Fan S. Centola M. Wu C.Y. Yamada N. El Gabalawy H. O'Shea J.J. J. Immunol. 2000; 164: 4659-4664Crossref PubMed Scopus (158) Google Scholar, 29Fukao T. Frucht D.M. Yap G. Gadina M. O'Shea J.J. Koyasu S. J. Immunol. 2001; 166: 4446-4455Crossref PubMed Scopus (160) Google Scholar). IL12-induced STAT4 activation in both B and NK cells stimulates IFNγ secretion. Immature DC and resting monocytes or macrophages express little STAT4. Upon DC maturation or monocyte/macrophage activation, for example by LPS, STAT4 expression is up-regulated and mediates IL12- or IFNα-induced IFNγ secretion. Human monocytes activate STAT4 in response only to IFNα, and not IL12 (28Frucht D.M. Aringer M. Galon J. Danning C. Brown M. Fan S. Centola M. Wu C.Y. Yamada N. El Gabalawy H. O'Shea J.J. J. Immunol. 2000; 164: 4659-4664Crossref PubMed Scopus (158) Google Scholar). Two reports have suggested that STAT4 may be expressed in non-lymphoid or non-myeloid cells. Specifically, STAT4 tyrosine phosphorylation was induced by cell surface binding of sublytic terminal complement complex in human aortic EC (30Niculescu F. Soane L. Badea T. Shin M. Rus H. Immunopharmacology. 1999; 42: 187-193Crossref PubMed Scopus (30) Google Scholar), and by treatment of human vascular smooth muscle cells (VSMC) with urokinase (31Dumler I. Kopmann A. Wagner K. Mayboroda O.A. Jerke U. Dietz R. Haller H. Gulba D.C. J. Biol. Chem. 1999; 274: 24059-24065Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). There are also some reports that both EC and VSMC may respond to IL12. In one study HUVEC were pretreated with IL12 prior to co-culture with allogeneic CD4+ T lymphocytes, and in the presence of the polyclonal mitogen phytohemagglutinin (PHA), IL12-treated EC enhanced IFNγ production by the T cells, compared with untreated EC (32Briscoe D.M. Henault L.E. Geehan C. Alexander S.I. Lichtman A.H. J. Immunol. 1997; 159: 3247-3256PubMed Google Scholar). A second study demonstrated that primary VSMC cultures treated with the combination of IL12 and IL18 could secrete IFNγ (33Gerdes N. Sukhova G.K. Libby P. Reynolds R.S. Young J.L. Schonbeck U. J. Exp. Med. 2002; 195: 245-257Crossref PubMed Scopus (437) Google Scholar). However, it is not known whether either IL12 or IFNα can activate a functional STAT4 signaling pathway in either human EC or VSMC, or whether STAT4 activation leads to the induction of gene expression in either cell type. In this report we demonstrate that human EC do express STAT4, but not the IL12 receptor. IFNα induces STAT4 tyro-sine phosphorylation, as does IL12 in HUVEC transduced to express both subunits of the IL12R. STAT4 signaling results in induction of the genes encoding the chemokine MCP1 and the suppressor of cytokine signaling (SOCS)3, identifying a STAT4-dependent pathway that may influence the pro-inflammatory functions of EC. Cytokines and Antibodies—Recombinant human IL12, IL6, IFNα2a, vascular endothelial cell growth factor (VEGF), and oncostatin M (OnM) were purchased from R&D Systems (Minneapolis, MN) and IFNγ from Biosource International (Camarillo, CA). Affinity-purified goat antibody to STAT4 was purchased from R&D Systems. Rabbit polyclonal antibody to phosphotyrosine-STAT4 was purchased from Zymed Laboratories (San Francisco, CA), and to STAT1, phosphotyrosine-STAT1, STAT3, and phosphotyrosine-STAT3 from Cell Signaling Technology (Beverly, MA), and rabbit polyclonal anti-SOCS3 antibody from IBL Laboratories (Fujioka-Shi, Japan). Mouse monoclonal antibody to β-actin was purchased from Sigma. Mouse monoclonal anti-IL12Rβ1 and rat monoclonal anti-IL12Rβ2 and isotype IgG controls were purchased from BD Biosciences (San Jose, CA). Horseradish peroxidase-conjugated donkey anti-goat, mouse, and rabbit antibodies, and phycoerythrin (PE) and fluorescein isothiocyanate-conjugated donkey anti-goat, mouse, rat, and rabbit antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). Cell Culture—All human cell types were isolated following protocols approved by the Yale Human Investigation Committee. Human umbilical vein EC (HUVEC) were isolated from discarded umbilical cords as previously described (34Gimbrone Jr., M.A. Prog. Hemost. Thromb. 1976; 3: 1-28PubMed Google Scholar). ECs from 2 or 3 cords were pooled and serially cultured at 37 °C in 5% CO2-humidified air on tissue culture plastic coated with 0.1% gelatin in Medium 199 (M199) containing 20% fetal calf serum (FCS), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine (all from Invitrogen, Carlsbad, CA), 50 μg/ml endothelial cell growth supplement (ECGS, BD Biosciences) and 100 μg/ml porcine intestinal heparin (Sigma). Confluent cultures were serially passaged by trypsinization and used for experiments at the second or third subculture. The culture medium was changed at least 24 h prior to any cytokine treatment. Cytokines were added directly to the cultures without further change of media. Human dermal microvascular endothelial cells (HDMEC) were isolated from discarded human skin, purified using anti-CD31 mini MACS beads (Miltenyi Biotec, Auburn, CA) and cultured in EGM2-MV growth medium (Clonetics, San Diego, CA) as previously described (35Kluger M.S. Johnson D.R. Pober J.S. J. Immunol. 1997; 158: 887-896PubMed Google Scholar). Human aortic and coronary artery VSMCs were isolated as previously described (36Mahboubi K. Kirkiles-Smith N.C. Karras J. Pober J.S. J. Biol. Chem. 2003; 278: 25014-25023Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), and cultured in M199 containing 20% FCS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2mmol/liter l-glutamine. Peripheral blood mononuclear cells (PBMCs) were obtained by leukapheresis of adult volunteer donors, and further purified by centrifugation over lymphocyte separation medium (Organon Teknika, Durham, NC) according to the manufacturer's instructions. Isolated PBMC were cultured in RPMI 1640 medium containing 10% FCS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mmol/liter l-glutamine. To produce PHA-activated PBMC, 5 × 106 PBMC were cultured in 1 μg/ml PHA for 3 days, supplemented with 10 units/ml IL2 (R&D systems) on day 2. The Phoenix-Ampho packaging cell line (a kind gift of Dr G. Nolan, Stanford University, Stanford, CA), and U3A cells (a kind gift of Dr. G. Stark, Cleveland Clinic Foundation) were cultured in Dulbecco's modified Eagles medium (Invitrogen) containing 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine. DNA Constructs and Retroviral Transduction—cDNAs encoding human IL12Rβ1 and IL12Rβ2 subcloned into the vectors pBluescript II SK+ and SK-, respectively, were purchased from ATCC (Manassas, VA). The coding sequences flanked by HindIII (5′) and NotI (3′) sites were amplified using the primers 5′-CCCAAGCTTGGGATAAGAAAGCGGCCATGGAGCCGCTGGTGACC-3′ and 5′-ATAGTTTAGCGGCCGCATTCTTATTCACATCTTGGCCTTGC-3′ for IL12Rβ1 and 5′-CCCAAGCTTGGGATAAGAAAGCGGCCATGGCACATACTTTTAGAGG-3′ and 5′-ATAGTTTAGCGGCCGCATTCTTATTCAGAGCATGAGGGAGTC-3′ for IL12Rβ2 (enzyme sites underlined, start and stop codons in bold), and subcloned into the LZRSpBMN-Z retroviral vector. Primers to amplify the coding region of STAT4 from a plasmid kindly provided by Dr. D. Farrar (Southwestern Medical Center, Dallas, TX) were 5′-CCCAAGCTTGGGATAAGAAAGCGGCCATGTCTCAGTGGAATCAAG-3′ and 5′-ATAGTTTAGCGGCCGCATTCTTATTCATTCAGCAGAATATGG-3′. This too was subcloned into LZRSpBMN-Z. STAT1 and, as a control, enhanced green fluorescent protein (EGFP) in the same vector were kindly provided by Dr. K. Mahboubi and Dr. A. Bothwell (Yale University, New Haven, CT). LZRSpBMN-Z constructs were transfected into Phoenix-Ampho cells using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Puromycin-resistant cells were selected and used to condition either M199 (containing 10% FCS and 2 mm l-glutamine but without ECGS, heparin or antibiotics) for HUVEC transduction or Dulbecco's modified Eagle's medium containing 10% FCS and 2 mm l-glutamine for U3A cell transduction. Collection of virus-conditioned medium and transduction of primary HUVEC cultures were performed as previously described (36Mahboubi K. Kirkiles-Smith N.C. Karras J. Pober J.S. J. Biol. Chem. 2003; 278: 25014-25023Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). For transduction of U3A cells, the cells were grown in virus-conditioned medium containing 8 μg/ml polybrene (Sigma) for 24 h and then passaged once prior to use. Immunoblotting, Immunoprecipitation, and ELISA—Lysis of confluent U3A cell and HUVEC cultures grown in 6-well or 10-cm diameter tissue culture dishes (Falcon, BD Biosciences), sample preparation, and immunoblotting were performed exactly as described previously (36Mahboubi K. Kirkiles-Smith N.C. Karras J. Pober J.S. J. Biol. Chem. 2003; 278: 25014-25023Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). For immunoprecipitation, confluent cultures in 10-cm plates were lysed in 1 ml of ice-cold radioimmune precipitation assay buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mm pefabloc, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mg/ml benzamidine, 1 mm sodium orthovanadate, and 1 mm sodium fluoride) and clarified by centrifugation at 10,000 × g for 10 min. Lysates were precleared by incubation with 4 μg of goat IgG (R&D Systems) and 50 μl of washed protein G-Sepharose (Amersham Biosciences) for 1 h at 4 °C. Following brief centrifugation to remove the beads, the lysates were incubated with 4 μg of STAT4 antibody and 50 μl of washed protein G-Sepharose at 4 °C overnight. The Sepharose beads were recovered by brief centrifugation, washed five times in ice-cold PBS and resuspended in 50 μl of SDS-PAGE sample buffer as previously described (36Mahboubi K. Kirkiles-Smith N.C. Karras J. Pober J.S. J. Biol. Chem. 2003; 278: 25014-25023Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Antibodies were used for immunoblotting at the following concentrations: STAT4 0.2 μg/ml; phosphotyrosine STAT4 4 μg/ml; STAT1, STAT3, phosphotyrosine STAT1, and phosphotyrosine STAT3 were used at 1:1000 the concentration supplied by the manufacturer; β-actin at 0.1 μg/ml; and SOCS3 at 1 μg/ml. Secondary peroxidase-conjugated antibodies were used at a 1:5000 dilution, following the manufacturer's instructions. ELISA kit for MCP1 was purchased from Biosource International, and used exactly as described by the manufacturer. Preparation of RNA, cDNA, and Procedure for Quantitative PCR—RNA was isolated from confluent cultures using RNeasy mini kits (Qiagen, Valencia, CA) with on-column DNase treatment exactly following the manufacturer's instructions. cDNA was synthesized using Taqman RT reagents (Applied Biosystems), following the manufacturer's instructions. PCR for IL12Rβ1, IL12Rβ2, and β-actin was performed using Hotstart Taq polymerase (Qiagen) following the manufacturer's instructions. The primers used are shown in Table I. Quantitative real-time PCR (Q-PCR) for MCP1, TAP1, SOCS3, and GAPDH was performed exactly as described (36Mahboubi K. Kirkiles-Smith N.C. Karras J. Pober J.S. J. Biol. Chem. 2003; 278: 25014-25023Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), using primers shown in Table I. All samples were analyzed in triplicate, and the results expressed as fold induction compared with baseline.Table IPCR primers used in this studyTargetPrimers (5′ → 3′)IL12Rβ1CCTGAAAACCCCCCACAGGCTGAGGCATTGCCCCCAIL12Rβ2CTGCAAACTGGCCTGTATCAAGTGCTCCAATGATTCACTCCβ-ActinTGCTATCCCTGTACGCCTCTAGTACTTGCGCTCAGGAGGAMCP1CCCCAGTCACCTGCTGTTATGCTTCTTTGGGACACTTGCTSOCS3GGCCACTCTTCAGCATCTCATCGTACTGGTCCAGGAACTCTAP1CAAGAGCCACAGGTATTTGGACTGCAGCAGCTGTGATTTCGAPDHGAAGGTGAAGGTCGGAGTCGAAGATGGTGATGGGATTTC Open table in a new tab DNA Microarray Analysis—Fluorescently labeled probes were prepared from 50 μg of total RNA, isolated as above. cDNA was synthesized using SuperScript II RNase H- reverse transcriptase (Invitrogen) in a volume of 40 μl using the manufacturer's buffer but containing 0.5 mm dATP, dGTP, and dCTP, 0.2 mm dTTP and 0.2 mm aminoallyl dUTP (Amersham Biosciences). Once the reaction was complete, the template RNA was hydrolyzed by incubation with 50 mm EDTA and 100 mm sodium hydroxide at 65 °C for 20 min. Following extraction with phenol/chloroform the cDNA was concentrated to a volume of 6 μl using an Amicon YM30 centrifugal filter (Millipore, Bedford, MA) and incubated with 1 μl of 5 mg/ml Cy3 or Cy5 monoreactive dyes dissolved in Me2SO (Amersham Biosciences) and 0.7 μl of 1 m sodium bicarbonate for 2 h at room temperature in the dark. Unconjugated dye was removed by addition of 500 μl of water and concentration in a YM30 column, repeated three times. Cy3- and Cy5-labeled probes were combined and resuspended in a final volume of 15 μl of water. Hybridization was performed in 1× Denhardt's solution (Amersham Biosciences), 4× SSPE (Ambion, Austin, TX), 0.2%SDS, containing 120 μg/ml poly(dA), 240 μg/ml yeast tRNA, and 600 μg/ml human CotI DNA (all from Invitrogen) in a final volume of 26.4 μl. The probe mixture was heated to 94 °C for 2 min and then cooled to 55 °C before application to a DNA microarray comprising 16,000 70-mer oligonucleotide spots prepared by the Yale Keck Foundation Biopolymer Resource Laboratory. The probe was covered with a coverslip and incubated overnight at 55 °C in a water bath. After removing the coverslip the slide was washed in 2× SSC (Ambion)/0.1% SDS, 0.2× SSC/0.1% SDS (twice), and 0.2× SSC, each for 12 min at room temperature. The microarray was scanned by the Yale/Keck facility and analyzed using GenePix software (Axon Instruments, Union City, CA). Flow Cytometry—For cell surface immunostaining, HUVEC, or HDMEC were washed twice with Hanks-buffered saline solution (HBSS) and incubated with trypsin/EDTA for 1 min. Detached cells were collected by centrifugation at 1000 × g for 5 min, washed twice with ice-cold PBS containing 1% bovine serum albumin and 0.1% sodium azide (PBS/BSA), and incubated with either IL12Rβ1 or IL12Rβ2 antibodies or isotype controls at 2 μg/ml in PBS/BSA for 2 h at 4 °C. After two further washes cells were incubated with 2 μg/ml PE- and fluorescein isothiocyanate-conjugated secondary antibodies in PBS/BSA for 1 h at 4 °C, washed a further two times and analyzed on a FACSort using Cellquest software (BD Biosciences). For intracellular immunostaining of STAT4, cells were detached as above and washed twice with ice-cold PBS before fixation with 2% paraformaldehyde for 15 min at room temperature. After a further two washes in PBS, cells were permeabilized for 15 min in PERM buffer (PBS containing 0.1% saponin, 1% fetal bovine serum and 0.1% sodium azide), washed once in PERM buffer and incubated with 1 μg/ml anti-STAT4 or goat IgG (R&D systems). Further washes and incubation with secondary antibody, all in PERM buffer, and flow cytometry were carried out as above. IFNα, but Not IL12 or IFNγ, Induces STAT4 Tyrosine Phosphorylation in Cultured HUVEC—IL12 or IFNα-mediated polarization of T cell immune responses to a TH1 phenotype depends on activation of the transcription factor STAT4. To determine whether this pathway is active in EC, we first investigated whether HUVEC express STAT4, and whether treatment with either IL12 or IFNα could induce STAT4 tyrosine phosphorylation. Because of its low abundance, we were unable to detect STAT4 by direct immunoblotting. To increase sensitivity we used immunoprecipitation with an affinity-purified polyclonal antibody, followed by immunoblotting, either with the STAT4 antibody or a phosphotyrosine-specific (Tyr693) STAT4 antibody. Fig. 1A shows that STAT4 could be immunoprecipitated from both HUVEC and PBMC. Treatment of both cell types with IFNα resulted in tyrosine phosphorylation of STAT4, which was transient in HUVEC when compared with PBMC. In contrast, IL12 induced STAT4 tyrosine-phosphorylation only in PBMC and not in HUVEC (Fig. 1A). Phosphorylation of STAT4 in HUVEC is specific to type 1 IFN, and not induced by treatment with IFNγ (Fig. 1B), although both interferon types induce STAT1 tyrosine phosphorylation (Fig. 1B). Parallel experiments using HDMEC and VSMC demonstrated STAT4 expression and tyrosine phosphorylation by IFNα in HDMEC but not in aortic or coronary artery VSMC (data not shown). These results demonstrate that cultured EC express STAT4, and that there appears to be a STAT4 signaling pathway in EC that may be activated by IFNα. Basal expression of STAT4 in both resting lymphocytes and monocytes is up-regulated upon activation of T cells, for example by PHA (16Cho S.S. Bacon C.M. Sudarshan C. Rees R.C. Finbloom D. Pine R. O'Shea J.J. J. Immunol. 1996; 157: 4781-4789PubMed Google Scholar), and of monocytes by LPS or IFNγ (28Frucht D.M. Aringer M. Galon J. Danning C. Brown M. Fan S. Centola M. Wu C.Y. Yamada N. El Gabalawy H. O'Shea J.J. J. Immunol. 2000; 164: 4659-4664Crossref PubMed Scopus (158) Google Scholar). We next investigated whether STAT4 expression in HUVEC could be induced by similar treatments. Incubation of HUVEC with IFNα, LPS, IL6, oncostatin M, or VEGF for 24 or 72 h neither enhanced nor suppressed STAT4 expression (data not shown). However, 72 h treatment with IFNγ both reduced total STAT4 levels and attenuated STAT4 tyrosine phosphorylation in response to IFNα (Fig. 1C). IFNα-induced STAT1 phosphorylation was unaffected by IFNγ pretreatment. HUVEC Transduced to Express the IL12 Receptor Phosphorylate STAT4 and STAT3 in Response to IL12—HUVEC may be unable to respond to IL12 because they do not express one or both of the IL12R subunits, or because they lack downstream components of IL12R-mediated signaling. To investigate the first possibility, we performed RT-PCR on RNA extracted from HUVEC and PBMC. Both IL12Rβ1 and IL12Rβ2 mRNA could be detected in PBMC, but neither in HUVEC (Fig. 2). This analysis was repeated on 3 HUVEC cultures, each derived from 2 donors. In no case could either IL12R subunit be identified. Similarly, we could not identify mRNA for either IL12R subunit in isolates of cultured HDMEC (not shown). We also failed to find IL12R subunits by RT-PCR and flow cytometry on both HUVEC and HDMEC that were either untreated or treated with IFNγ, TNF, both in combination, LPS or IFNα (data not shown). We next determined whether IL12 could induce STAT4 tyrosine phosphorylation in HUVEC transduced to express the IL12 receptor. Primary cultures of HUVEC were transduced to express both IL12Rβ1 and IL12Rβ2 or, as a control, EGFP. Cell surface expression of each subunit was analyzed by flow cytometry after 4 rounds of retroviral transduction. More than 95% of the HUVEC expressed either IL12R subunit, and 84% expressed both (Fig. 3A). Comparable frequencies of EGFP expression were o
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