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Chemokine-Cytokine Cross-talk

THE ELR+ CXC CHEMOKINE LIX (CXCL5) AMPLIFIES A PROINFLAMMATORY CYTOKINE RESPONSE VIA A PHOSPHATIDYLINOSITOL 3-KINASE-NF-κB PATHWAY*
Open AccessPublished:December 04, 2002DOI:https://doi.org/10.1074/jbc.M207006200
      It is well established that cytokines can induce the production of chemokines, but the role of chemokines in the regulation of cytokine expression has not been fully investigated. Exposure of rat cardiac-derived endothelial cells (CDEC) to lipopolysaccharide-induced CXC chemokine (LIX), and to a lesser extent to KC and MIP-2, activated NF-κB and induced κB-driven promoter activity. LIX did not activate Oct-1. LIX-induced interleukin-1β and tumor necrosis factor-α promoter activity, and up-regulated mRNA expression. Increased transcription and mRNA stability both contributed to cytokine expression. LIX-mediated cytokine gene transcription was inhibited by interleukin-10. Transient overexpression of kinase-deficient NF-κB-inducing kinase (NIK) and IκB kinase (IKK), and dominant negative IκB significantly inhibited LIX-mediated NF-κB activation in rat CDEC. Inhibition of Gi protein-coupled signal transduction, poly(ADP-ribose) polymerase, phosphatidylinositol 3-kinase, and the 26 S proteasome significantly inhibited LIX-mediated NF-κB activation and cytokine gene transcription. Blocking CXCR2 attenuated LIX-mediated κB activation and κB-driven promoter activity in rat CDEC that express both CXCR1 and -2, and abrogated its activation in mouse CDEC that express only CXCR2. These results indicate that LIX activates NF-κB and induces κB-responsive proinflammatory cytokines via either CXCR1 or CXCR2, and involved phosphatidylinositol 3-kinase, NIK, IKK, and IκB. Thus, in addition to attracting and activating neutrophils, the ELR+ CXC chemokines amplify the inflammatory cascade, stimulating local production of cytokines that have negative inotropic and proapoptotic effects.
      Chemokines are small molecular weight cytokines involved in activation of specific subsets of immune cells and their recruitment to the site of injury and inflammation (
      • Oppenheim J.J.
      • Zachariae C.O.
      • Mukaida N.
      • Matsushima K.
      ,
      • Baggiolini M.
      • Dewald B.
      • Moser B.
      ,
      • Rollins B.J.
      ,
      • Schall T.J.
      • Bacon K.B.
      ,
      • Rovai L.E.
      • Herschman H.R.
      • Smith J.B.
      ,
      • Maekawa T.
      • Ishii T.
      ,
      • Simonini A.
      • Moscucci M.
      • Muller D.W.
      • Bates E.R.
      • Pagani F.D.
      • Burdick M.D.
      • Strieter R.M.
      ). They are classified into C, CC, CXC, and CX3C families (
      • Baggiolini M.
      • Dewald B.
      • Moser B.
      ,
      • Rollins B.J.
      ,
      • Schall T.J.
      • Bacon K.B.
      ). In the CXC family, the first two conserved cysteines are separated by one nonconserved amino acid (X in CXC). CXC chemokines that have a glutamic acid-leucine-arginine (ELR) sequence immediately preceding the CXC motif are potent neutrophil chemoattractants (ELR+ CXC chemokines) (
      • Baggiolini M.
      • Dewald B.
      • Moser B.
      ,
      • Lukacs N.W.
      • Hogaboam C.
      • Campbell E.
      • Kunkel S.L.
      ,
      • Kukielka G.L.
      • Smith C.W.
      • LaRosa G.L.
      • Manning A.M.
      • Mendoza L.H.
      • Daly T.J.
      • Hughes B.J.
      • Youker K.A.
      • Hawkins H.K.
      • Michael L.H.
      • Rot A.
      • Entman G.L.
      ). Neutrophil migration is stimulated by a gradient of these chemokines from blood toward the site of inflammation.
      We have recently shown up-regulation of the ELR+ CXC chemokines, LIX
      The abbreviations used are: LIX, lipopolysachharide-induced CXC chemokine; CDEC, cardiac-derived endothelial cells; IL, interleukin; EMSA, electrophoretic mobility shift assay; IKK, IκB kinase; KC, cytokine-induced neutrophil chemoattractant; MIP-2, macrophage inflammatory protein-2; NIK, NF-κB inducing kinase; PARP-1, poly(ADP-ribose) polymerase; PI3K, phosphatidylinositol 3-kinase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PKC, protein kinase C; EGFP, enhanced green fluorescent protein; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; TNF-α, tumor necrosis factor-α; dn, dominant negative; kd, kinase-deficient
      1The abbreviations used are: LIX, lipopolysachharide-induced CXC chemokine; CDEC, cardiac-derived endothelial cells; IL, interleukin; EMSA, electrophoretic mobility shift assay; IKK, IκB kinase; KC, cytokine-induced neutrophil chemoattractant; MIP-2, macrophage inflammatory protein-2; NIK, NF-κB inducing kinase; PARP-1, poly(ADP-ribose) polymerase; PI3K, phosphatidylinositol 3-kinase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PKC, protein kinase C; EGFP, enhanced green fluorescent protein; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; TNF-α, tumor necrosis factor-α; dn, dominant negative; kd, kinase-deficient
      (lipopolysaccharide-induced CXC chemokine; CXCL5), KC (cytokine-induced neutrophil chemoattractant; CXCL1), and MIP-2 (macrophage inflammatory protein-2; CXCL2) in a rat model of myocardial ischemia/reperfusion injury (
      • Chandrasekar B.
      • Smith J.B.
      • Freeman G.L.
      ). High levels of myocardial neutrophil infiltration coincided with peak levels of LIX and MIP-2 expression. Neutralization of LIX, KC, and MIP-2 inhibited myeloperoxidase activity, a measure of neutrophil infiltration, by 79, 28, and 37%, respectively, indicating that LIX may be the predominant neutrophil chemoattractant in this model of reperfusion injury (
      • Chandrasekar B.
      • Smith J.B.
      • Freeman G.L.
      ). Furthermore, the proinflammatory cytokine expression preceded the chemokine expression in this model, suggesting that chemokine expression was a downstream effect of cytokine production (
      • Chandrasekar B.
      • Smith J.B.
      • Freeman G.L.
      ). This was confirmed in in vitro studies where exposure of cardiomyocytes to TNF-α induced LIX expression via NF-κB activation (
      • Chandrasekar B.
      • Smith J.B.
      • Freeman G.L.
      ).
      NF-κB is a ubiquitous, multisubunit, inducible transcription factor that regulates the expression of various genes involved in the immune and inflammatory processes (
      • Bowie A.
      • O'Neill L.A.J.
      ,
      • Mercurio F.
      • Manning A.M.
      ). The p50/p65 heterodimer, which has been most studied, resides in the cytoplasm in an inactive state because of binding of p65 to an inhibitory subunit IκB. The IκB family, including IκB-α, IκB-β, IκB-γ, IκB-ε, all prevent activation and subsequent nuclear translocation of the heterodimer. Various stimuli including cytokines, growth factors, and oxidative stress induce IκB hyperphosphorylation leading to its selective degradation in the cytoplasm by the ubiquitin-26 S proteasome system, resulting in NF-κB activation (
      • Bowie A.
      • O'Neill L.A.J.
      ,
      • Mercurio F.
      • Manning A.M.
      ).
      A multiprotein complex comprised of IKK (IκB kinase)-α, IKK-β, and a regulatory subunit IKK-γ/NEMO was shown to mediate phosphorylation of IκB by various cytokines (
      • Mercurio F.
      • Zhu H.
      • Murray B.W.
      • Shevchenko A.
      • Bennett B.L.
      • Li J.
      • Young D.B.
      • Barbosa M.
      • Mann M.
      • Manning A.
      • Rao A.
      ,
      • Stancovski I.
      • Baltimore D.
      ,
      • Cohen L.
      • Henzel W.J.
      • Baeuerle P.A.
      ,
      • Rothwarf D.M.
      • Zandi E.
      • Natoli G.
      • Karin M.
      ,
      • Zandi E.
      • Rothwarf D.M.
      • Delhase M.
      • Hayakawa M.
      • Karin M.
      ). The cytokine-initiated signal transduction cascade leading to IκB phosphorylation has been shown to converge at activation of the IKK by NF-κB-inducing kinase (NIK). NIK associates with IKK-γ and activates the IKK signalsome. PI 3-kinase, PI-phospholipase C, protein kinase C, and p38 mitogen-activated protein kinase were implicated as upstream regulators of NIK and IKK. Furthermore, poly(ADP-ribose) polymerase 1 (PARP-1), a nuclear protein involved in DNA repair, has been shown to physically and functionally associate with NF-κB in the nucleus and modulate NF-κB-dependent cytokine gene transcription (
      • Ha H.C.
      • Hester L.D.
      • Snyder S.H.
      ,
      • Hassa P.O.
      • Covic M.
      • Hasan S.
      • Imhof R.
      • Hottiger M.O.
      ). The role of these various regulatory subunits in chemokine-mediated NF-κB activation and cytokine gene transcription has not been investigated.
      Whereas the agonistic effects of cytokines on chemokine expression are well described, very little is known about chemokine-mediated cytokine expression. In the present study we investigated the role of the ELR+ CXC chemokines, LIX, KC, and MIP-2, in NF-κB activation and induction of IL-1β and TNF-α expression. Furthermore, we explored the chemokine receptor usage and signal transduction pathway involved in chemokine-mediated NF-κB activation. Our results indicate that the ELR+ CXC chemokines activate NF-κB, induce proinflammatory cytokine expression, and signal through CXCR2, and presumably also through CXCR1.

      EXPERIMENTAL PROCEDURES

      Materials and Reagents

      Recombinant mouse LIX, KC, and MIP-2 were obtained from PeproTech, Inc. (Rocky Hill, NJ). Recombinant carrier-free rat IL-1β and rat TNF-α were from R&D Systems (Minneapolis, MN). The recombinant proteins contained <1 ng of endotoxin per μg of protein. Polyclonal antibodies against rat IL-1β and TNF-α were fromBIOSOURCE International (Camarillo, CA), and IκB-α, anti-p50 (sc-1114X), and anti-p65 (sc-372X) subunit-specific polyclonal antibodies, and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology, Inc. Phospho-IκB-α (Ser32) polyclonals, which detect only the phosphorylated form of IκB-α, and not the nonphosphorylated form, were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Normal rabbit IgG (control IgG) was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Anti-FLAG, anti-Myc, and anti-HA antibodies were from Sigma, Roche Applied Biosciences (Indianapolis, IN), and Covance Inc. (Princeton, NJ), respectively. All tissue culture supplies were from Invitrogen. Radiochemicals ([α-32P]dCTP, [γ-32P]ATP, and [α-32P]UTP) were purchased from Amersham Biosciences. SB 447232 (N-[2-hydroxy-3-(N“-isoxazolidinyl sulfonamide)-4-chlorophenyl)-N′-(2,3-dichlorophenyl)urea) was synthesized in the Department of Medicinal Chemistry at GlaxoSmithKline (King of Prussia, PA). Wortmannin, LY 294002, chelerythrine chloride, MG-132, 3-aminobenzamide, and pertussis toxin were obtained from Calbiochem (San Diego, CA). All other chemicals were purchased from Sigma.

      Cell Culture

      Nontransformed rat cardiac-derived endothelial cells (rat CDEC; a generous gift of C. A. Diglio; Ref.
      • Diglio C.A.
      • Grammas P.
      • Giacomelli F.
      • Wiener J.
      ) and nontransformed mouse cardiac-derived endothelial cells, described previously (
      • Marelli-Berg F.M.
      • Peek E.
      • Lidington E.A.
      • Stauss H.J.
      • Lechler R.I.
      ), were cultured in medium 199 with 10% fetal calf serum, endothelium growth supplement (30 mg/liter), heparin (100 mg/liter), penicillin (100,000 units/liter), and streptomycin (100 mg/liter) at 37 °C in a humidified atmosphere of 95% air, 5% CO2. At 70–80% confluency, the media was replaced with serum-free medium 199 containing 0.5% BSA. After overnight culture, LIX, KC, MIP-2, or PBS was added and incubated for the indicated time periods. To inhibit NF-κB DNA binding activity, the cells were pretreated for 1 h with 3-aminobenzamide (10 mm in ethanol), wortmannin (50 nm), LY 294002 (20 μm), chelerythrine chloride (60 μm), and MG-132 (5 μm) in Me2SO, pertussis toxin (100 ng/ml) in PBS or for 4 h with IL-10 (10 ng/ml) or corresponding vehicle before the addition of LIX.

      Transient Cell Transfections and Reporter Assays

      The NF-κB driven luciferase reporter plasmid (pNF-κB-Luc) was obtained from Stratagene (La Jolla, CA) and contains five copies of NF-κB consensus sequence linked to the minimal E1B promoter-luciferase reporter gene. pEGFP-Luc was used as a control. The phosphorylation-deficient S32A/S36A mutant of IκB-α (pCMX-IκB-α(S32A/S36A)) was a gift from Inder Verma (The Salk Institute, La Jolla, CA), and the Myc-tagged phosphorylation-deficient S19A/S32A mutant of IκB-β in pCMV-Tag3B (Stratagene) has been described earlier (
      • Dulin N.O.
      • Niu J.
      • Browning D.D.
      • Ye R.D.
      • Voyno-Yasenetskaya T.
      ). Kinase-deficient NIK (pRK7-NIK(KK429–430AA)-Flag), IKK-β (pRK5-IKK-β-Flag), and dominant negative IKK-γ (pcDNA3-IKK-γ-HA) were obtained from David V. Goeddel (Tularik Inc., South San Francisco, CA), Tom Maniatis (Harvard University, Cambridge, MA), and Gabriel Nunez (University of Michigan Medical School, Ann Arbor, MI), respectively. Rat CDEC were plated on six-well tissue culture dishes and transfected the following day at ∼70–80% confluency using LipofectAMINE 2000TM(Invitrogen, Carlsbad, CA) as described by the manufacturer. pRLRenilla-luciferase reporter gene (100 ng; pRL-TK vector;Promega, Madison, WI) was used as an internal control. The empty vectors pCMX, pCMV-Tag3B, pRK5, pRK7, and pcDNA3 were used as controls. Data were normalized for transfection efficiency by dividing firefly luciferase activity with that of correspondingRenilla luciferase, and expressed as mean relative stimulation ± S.E. for a representative experiment from three separate experiments, each performed in triplicate. The amount of DNA transfected was kept constant (2 μg) in all transfection experiments. After transfection, the cells were found to be viable (trypan blue dye exclusion). 24 h after transfection, the media was changed, and the cells were exposed to LIX, KC, or MIP-2 at the indicated concentrations and for the specified time periods. Cell extracts were prepared, and luciferase activity was determined with a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA) using the PromegaBiotechTM dual-luciferase reporter assay system.
      Transfection efficiency was determined by transfecting rat CDEC with pEGFP-N1 vector (Clontech, Palo Alto, CA) that constitutively expresses the enhanced green fluorescent protein (EGFP) under the regulation of CMV promoter and enhancer. Once the cells reached ∼70% confluency, the media was replaced with M199 + 0.5% BSA. After overnight culture, cells were transfected with pEGFP-N1 and LipofectAMINE 2000. 24 h later, the cells were trypsinized, seeded onto Lab-Tek II chamber slide (NuncTM), and cultured for an additional 48 h. Cells were then washed in PBS (pH 7.4), fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. After washing in PBS, coverslips were mounted using ProLongTMAntifade kit (Molecular Probes, Eugene, OR). After the mounting media was dried, the coverslips were sealed with black nail polish, and stored at 4 °C in the dark. The cells were visualized by a fluorescent microscope (Nikon Eclipse TE200, Nikon Inc., Melville, MA), and 1,000 cells were counted under ×20 objective, and bright to very-bright green fluorescent cells were considered positive for the expression of EGFP, and the others as nontransfected (controls). The transfection efficiency varied between 37 and 46% with an average of 38.1 ± 2.9%. To determine the role of CXC receptors in LIX-mediated NF-κB activation, rat and mouse CDEC were treated with SB447232 (GlaxoSmithKline Beecham), a specific CXCR2 antagonist, for 10 min before the addition of LIX.

      Reverse Transcriptase-Polymerase Chain Reaction

      To demonstrate expression of CXCR1 and CXCR2, reverse transcriptase-PCR was performed using total RNA isolated from rat CDEC. The primers were designed based on published sequences for CXCR1 and CXCR2 in rats (
      • Nguyen D.
      • Stangel M.
      ,
      • Dunstan C.A.
      • Salafranca M.N.
      • Adhikari S.
      • Xia Y.
      • Feng L.
      • Harrison J.K.
      ,
      • Gobl A.E.
      • Huang M.R.
      • Wang S.
      • Zhou Y.
      • Oberg K.
      ). In brief, total RNA was isolated with lysis buffer containing phenol and guanidine isothiocyanate (TRIzol reagent, Invitrogen). 2 μg of total RNA was reverse transcribed into cDNA with Moloney murine leukemia virus-reverse transcriptase (Invitrogen) and random hexamers. Amplification of CXCR1 (183 bp) and CXCR2 (413 bp) cDNAs was performed using the following primers: CXCR1-sense, 5′-CAGGCTTCTCCAGCACACAAG-3; CXCR1-antisense, 5′-TTGGTCATTGGAACCCTCTTAC-3′; and CXCR2-sense, 5′-GCAAACCCTTCTACCGTAG-3; CXCR2-antisense, 5′-AGAAGTCCATGGCGAAATT-3′. Amplification was performed with an initial denaturation at 94 °C for 1 min, followed by 35 cycles of 94 °C, 30 s; 52 °C, 30 s; 72 °C, 1 min with a final 7-min extension. The PCR products were electrophoresed at 100 volts on a Tris-acetate-EDTA, 2% agarose gel containing ethidium bromide.

      Electrophoretic Mobility Shift Assay

      NF-κB DNA binding activity was measured in the nuclear protein extracts by electrophoretic mobility shift assay (EMSA) as described earlier (
      • Chandrasekar B.
      • Smith J.B.
      • Freeman G.L.
      ,
      • Chandrasekar B.
      • Mitchell D.H.
      • Colston J.T.
      • Freeman G.L.
      ). In the gel supershift assay, the protein extract (10 μg) was preincubated for 40 min on ice with either anti-p50 or -p65 subunit-specific polyclonal antibodies (1 μg) or control IgG (1 μg) prior to the addition of 32P-labeled double stranded NF-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′). Absence of protein extract, competition with 100-fold molar excess unlabeled consensus NF-κB, and mutant NF-κB oligonucleotide (5′-AGTTGAGGCGACTTTCCCAGGC-3′; Santa Cruz Biotechnology, Inc.) served as controls. Levels of Oct-1, a constitutively expressed transcription factor, were also measured by EMSA using Oct-1 consensus sequence (5′-TGTCGAATGCAAATCACTAGAA-3′; Santa Cruz Biotechnology, Inc.).

      Northern Blot Analysis

      Total cellular RNA was isolated using the TRIzol reagent (Invitrogen). 20 μg of total RNA were resolved on a 0.8% agarose-formaldehyde gel and electroblotted onto nitrocellulose membrane. After prehybridization for 4 h, hybridizations were carried out at 42 °C for 16 h, followed by high stringency washing at 68 °C in 0.1× SSC, 0.1% SDS. The cDNAs were amplified using total RNA isolated from rat CDEC and gene-specific primers (rat IL-1β, GenBankTM accession number NM_031512, 324-bp product, sense, 5′-CTCTGTGACTCGTGGGATGATGAC-3′ (bases 383–405) and antisense, 5′-TCTTCTTCTTTGGGTATTGTTTGG-3′ (bases 684–707); TNF-α, GenBankTM accession number AF329985, 295-bp product, sense, 5′-TACTGAACTTCGGGGTGATTGGTCC-3′ (bases 955–979) and antisense, 5′-CAGCCTTGTCCCTTGAAGAGAACC-3′ (bases 2161–2138). The PCR products were cloned into pCRTM2.1-TOPOTM vector (Invitrogen) and sequenced on both strands for confirmation. The probe for rat LIX (GenBankTM accession number U90448) was a 329-bp cDNA cloned into pCRTM2.1-TOPO vector from a reverse transcriptase-PCR product generated with primers: sense, 5′-GGTCCTGCTCGTCATTCA-3′ (bases 41 to 58) and antisense, 5′-CAGTGCAAGTGCATTCCGCT-3′ (bases 350 to 369). The cDNAs were labeled with [α-32P]dCTP (3,000 Ci/mmol; AmershamBiosciences) using random hexanucleotide primers (Roche Molecular Biochemicals, Indianapolis, IN). Expression levels were normalized to 28 S rRNA expression. The 28 S rRNA probe (40 base single stranded oligonucleotide; Oncogene Science, Uniondale, NY) was 5′ end-labeled with [γ-32P]ATP using T4 polynucleotide kinase (
      • Chandrasekar B.
      • Mitchell D.H.
      • Colston J.T.
      • Freeman G.L.
      ).

      Interleukin-1β and TNF-α Promoter Analyses

      Murine IL-1β Promoter

      The murine IL-1β promoter (−4093 to +45) construct in pBluescript vector was a kind gift from Clifford J. Bellone (St. Louis University School of Medicine, St. Louis, MO; Ref.
      • Godambe S.A.
      • Chaplin D.D.
      • Takova T.
      • Bellone C.J.
      ). This construct contains 4,093 bp of the 5′-flanking sequence that includes the first exon, first intron, and untranslated region of the second exon. This promoter construct has been demonstrated to confer a strong responsiveness to lipopolysaccharide (
      • Godambe S.A.
      • Chaplin D.D.
      • Takova T.
      • Bellone C.J.
      ). Rat CDEC were transfected with 3 μg of either the IL-1β −4093 to +45-CAT (chloramphenical acetyltransferase) or a mock plasmid that contains CAT reporter gene alone (pFR-CAT; Stratagene). To compensate for variations in transfection, cells were cotransfected with a β-galactosidase reporter construct (pSV-β-galactosidase control vector, Promega) in which the SV40 early promoter and enhancer drives transcription of thelacZ gene, which encodes the β-galactosidase enzyme. 24 h later, the media was changed, and the cells were treated with LIX (100 ng/ml), neutralized LIX, or vehicle. Seven hours later the cells were processed for CAT levels using a CAT enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals) and β-galactosidase levels by using the β-galactosidase assay kit (Invitrogen) essentially as described by the manufacturers.

      Murine TNF-α Promoter

      Rat CDEC were transfected with pTNF−1080/+138, pTNF−85/+135, or empty vector (pGL3 basic). These murine TNF-α promoter reporter constructs were described earlier (
      • Singh I.S.
      • Viscardi R.M.
      • Kalvakolanu I.
      • Calderwood S.
      • Hasday J.D.
      ). The 1.1-kb TNF-α promoter construct (−1080/+138 nucleotides; relative to the transcription start site) contains 4 NF-κB response elements, and has been demonstrated to confer responsiveness to a variety of stimuli including lipopolysaccharide. Its deletion mutant construct (−85/+135) lacks all the four NF-κB response elements but contains only TATA box and Sp1 site, and responds poorly to lipopolysaccharide (
      • Singh I.S.
      • Viscardi R.M.
      • Kalvakolanu I.
      • Calderwood S.
      • Hasday J.D.
      ). The cells were co-transfected with pRK-Renilla to compensate for transfection efficiency. 24 h after transfection, the media was changed, and the cells were treated with LIX, neutralized LIX, or vehicle. Seven hours later the cells were processed for luciferase activity by the dual luciferase assay kit.

      mRNA Stability-Actinomycin D Pulse

      Rat CDEC were cultured in M199 medium containing 10% fetal calf serum. At 70–80% confluency, the complete medium was replaced with M199 + 0.5% BSA, and cultured for an additional 16 h. The cells were then treated with either LIX (100 ng/ml) or vehicle (control) for 4 h. Actinomycin D (5 μg/ml; Sigma), a potent inhibitor of RNA polymerase II-dependent transcription, was then added. At the indicated time periods 1.5, 3, 4.5, and 6 h), cells were harvested for total RNA isolation. RNA was isolated using TRIzol reagent, and analyzed by Northern blot hybridization to quantitate IL-1β and TNF-α mRNA levels as described above.

      LIX-mediated IL-1β and TNF-α Transcription (Nuclear Run-on)

      After treating rat CDEC with LIX (100 ng/ml) for 4 h in M199 medium containing 0.5% BSA, nuclei were isolated, counted in a hemocytometer, and resuspended (2 × 108/ml) in a storage buffer (50 mm Tris-HCl, pH 8.0, 5 mmMgCl2, 0.1 mm EDTA, 2 mmdithiothreitol, 40% glycerol) as described in detail previously (
      • Clark R.A.
      • Li S.L.
      • Pearson D.W.
      • Leidal K.G.
      • Clark J.R.
      • Denning G.M.
      • Reddick R.
      • Krause K.H.
      • Valente A.J.
      ). The nuclei were aliquoted and snap frozen in methanol/dry ice bath and stored in liquid N2 until further use. For labeling RNA, nuclei were thawed (100 μl), mixed with equal volumes of labeling mixture (200 mm KCl, 8 mm MgCl2, 1 mm each of ATP, UTP, and CTP, 100 μm GTP) and 100 μCi of [α-32P]UTP (800 Ci/mmol). The mixture was incubated at 30 °C for 30 min and 20 μl (4 μg) of RQ1 RNase-free DNase I (Promega Corp.) was added and incubated for an additional 10 min. After digestion with proteinase K (60 μg in 20 μl) in a buffer containing 1% SDS, 50 mm Tris-HCl (pH 7.0), 50 mm EDTA for 30 min at 42 °C, it was subjected to phenol/chloroform/isoamyl alcohol and chloroform extractions. The aqueous phase was ethanol-precipitated in the presence of 20 μg of carrier RNA (Escherichia coli transfer RNA, RNase-free, Roche Molecular Biochemicals). The pellet was dissolved in 80 μl of STE buffer (100 mm NaCl, 20 mm Tris-HCl, pH 7.5, 10 mm EDTA), and the unincorporated label was removed using NucTrapTM probe purification columns (Stratagene), and the incorporated radioactivity was determined in a scintillation counter.
      Equal amounts of plasmid vectors containing IL-1β, TNF-α, glyceraldehyde-3-phosphate dehydrogenase (GenBankTMaccession number M17701; 339-bp product, sense: 5′-TCCGCCCCTTCCGCTGATG-3′ (bases 388–406), antisense: 5′-CACGGAAGGCCATGCCAGTGA-3′ (bases 707–727)), or empty plasmid (pCRTM2.1-TOPOTM vector) were alkaline denatured, applied to nitrocellulose membranes using a slot-blot apparatus (HYBRI-SLOTTM MANIFOLD, Invitrogen). After fixing the DNA to the membranes by UV cross-linking, prehybridization was performed at 52 °C overnight, followed by hybridization for 3 days with ∼106 cpm/ml of labeled RNA. The filters were then washed three times for 10 min each in 2× SSC plus 0.1% SDS, two times in 0.1× SSC plus 0.1% SDS at 65 °C for 15 min each, and then treated with RNase A (10 mg/ml) for 30 min at 37 °C in 2× SSC. Finally the membranes were washed with 2× SSC at 37 °C for 30 min, and subjected to autoradiography, and the visualized bands were semiquantitated by densitometry.

      Protein Extraction and Western Blot Analysis

      30 μg of cell extract in RIPA buffer (50 mmTris-HCl, pH 7.4, 150 mm NaCl, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml each of aprotinin, leupeptin, and pepstatin, 1% Nonidet P-40, 1 mmsodium orthovanadate, and 1 mm NaF) from untreated (control) and treated CDEC were subjected to SDS-PAGE under reducing conditions, and electrotransferred onto polyvinylidene difluoride membranes (Millipore, MA). Nonspecific sites were blocked with 10% normal goat serum (preimmune; DAKO) for 1 h at room temperature, drained, and incubated overnight at 4 °C with the primary antibody in TBST (Tris-buffered saline containing 0.5% Tween 20) containing normal goat serum, washed in TBST, and incubated further for 1 h with the secondary antibody conjugated to horseradish peroxidase. After extensive washings with TBST, the membranes were incubated with an enhanced chemiluminescence reagent (Amersham Biosciences). The membranes were then washed, exposed to Kodak X-Omat AR film, and the autoradiographic bands were semiquantified and normalized to β-actin levels (
      • Chandrasekar B.
      • Smith J.B.
      • Freeman G.L.
      ,
      • Chandrasekar B.
      • Mitchell D.H.
      • Colston J.T.
      • Freeman G.L.
      ).

      Enzyme-linked Immunosorbent Assay

      TNF-α (sensitivity 0.7 pg/ml) and IL-1β (sensitivity <3.0 pg/ml) levels in culture supernatants were measured by enzyme-linked immunosorbent assay using commercially available kits (BIOSOURCE International; Ref.
      • Murray D.R.
      • Prabhu S.D.
      • Chandrasekar B.
      ). Studies were performed as per the manufacturer's instructions.

      Measurement of PI3K Activity

      PI3K lipid kinase assays were performed essentially as described by Foukas et al. (
      • Foukas L.C.
      • Daniele N.
      • Ktori C.
      • Anderson K.E.
      • Jensen J.
      • Shepherd P.R.
      ). After overnight incubation in 0.5% BSA, M199 media, rat CDEC were treated with rLIX (100 ng/ml) for 5 min with and without LY 294002. Cleared cell lysates were prepared by centrifugation at 10,000 × g for 30 min at 4 °C, and protein concentration was determined. Equal amounts of protein was immunoprecipitated with affinity purified antibodies against the p85 regulatory subunit of PI3K (Santa Cruz Biotechnology, Inc., number sc-423) for 2 h followed by protein A-Sepharose (AmershamBiosciences) for 1 h at 4 °C. After washing the immunoprecipitates (IP) in Tris-HCl (100 mm, pH 7.4) containing 0.5 m LiCl and kinase assay buffer (2×, 100 mm HEPES-NaOH, pH 7.4, 200 mm NaCl, 2 mm dithiothreitol), the immunoprecipitates were resuspended in 50 μl of 1× kinase assay buffer containing 5 mmMgCl2, 100 μm ATP (plus 0.1 μCi of [γ-32P]ATP/assay), and 200 μg/ml phosphatidylinositol as a substrate. The reaction was incubated at 25 °C for 20 min. The reaction was stopped by the addition of 100 μl of 0.1 mHCl and 200 μl of chloroform/methanol (1:1). The lower organic phase containing phospholipids was recovered and spotted on silica gel thin-layer chromatography plates (Gel-60, Merck), impregnated with 1% (w/v) potassium oxalate, 1 mm EDTA in water/methanol (6:4), and developed in a mixture of chloroform, methanol, 4 mNH3 (9:7:4). The radioactivity on the dried plate was visualized and quantified by autoradiography and densitometry.

      Measurement of Intracellular Calcium

      Intracellular calcium measurements were made in rat CDEC using the calcium-sensitive probe Fura-2/AM (Molecular Probes). The cells were loaded with Fura-2/AM pentapotassium salt (5 μm) in M199 medium supplemented with 10% fetal calf serum. After incubation for 45 min at 37 °C, the cells were washed and resuspended at 2 × 106 cells/ml in 137 mm NaCl, 4.5 mm KCl, 1.2 mmMgCl2·7H2O, 4.9 mm KCl, 1.2 mm NaH2PO4, 20 mmHEPES, 15 mmd-glucose, 1.8 mmCaCl2 (pH 7.4). The cell suspension was placed in a fluorimetry cuvette and stirred continuously at 37 °C. After equilibrating at 37 °C for 10 min, rLIX (100 ng/ml), neutralized LIX, or PBS were added, and fluorescence was monitored at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm on a Hitachi F-2000 fluorescence spectrophotometer; the results were calculated as the ratio of emission following excitation at 340 nm with that produced by excitation at 380 nm. The Fura-2/AM-loaded cells were treated with either Triton X-100 (1%) or 100 mm EGTA to obtain maximal and minimal fluorescence, and the data were normalized as a percentage of the maximal fluorescence.

      Inhibition of Radioligand Binding and Calcium Mobilization

      Procedures utilized for 125I-IL-8 binding to membranes of Chinese hamster ovary cells stably expressing CXCR1 and CXCR2 were done as previously described (
      • White J.R.
      • Lee J.M.
      • Young P.R.
      • Hertzberg R.P.
      • Jurewicz A.J.
      • Chaikin M.A.
      • Widdowson K.
      • Foley J.J.
      • Martin L.D.
      • Griswold D.E.
      • Sarau H.M.
      ,
      • Sarau H.M.
      • Widdowson K.L.
      • Palovich M.R.
      • White J.R.
      • Underwood D.C.
      • Griswold D.E.
      ). Inhibition of 10 nm IL-8-induced calcium mobilization in RBL 2H3 cells stably expressing CXCR1 or CXCR2 was done as previously described (
      • Sarau H.M.
      • Widdowson K.L.
      • Palovich M.R.
      • White J.R.
      • Underwood D.C.
      • Griswold D.E.
      ). Inhibition of calcium mobilization with human polymorphonuclear leukocytes was done using 1 nm IL-8 or 10 nm GROα as described (
      • White J.R.
      • Lee J.M.
      • Young P.R.
      • Hertzberg R.P.
      • Jurewicz A.J.
      • Chaikin M.A.
      • Widdowson K.
      • Foley J.J.
      • Martin L.D.
      • Griswold D.E.
      • Sarau H.M.
      ). In addition, the same procedure was used for inhibition of rat GROβ-induced calcium mobilization in rat polymorphonuclear leukocytes isolated from peripheral blood (
      • White J.R.
      • Lee J.M.
      • Young P.R.
      • Hertzberg R.P.
      • Jurewicz A.J.
      • Chaikin M.A.
      • Widdowson K.
      • Foley J.J.
      • Martin L.D.
      • Griswold D.E.
      • Sarau H.M.
      ,
      • Sarau H.M.
      • Widdowson K.L.
      • Palovich M.R.
      • White J.R.
      • Underwood D.C.
      • Griswold D.E.
      ).

      Statistical Analysis

      Comparisons between controls and various treatments were performed for measures of NF-κB DNA-binding activity, κB-driven luciferase activity, and cytokine mRNA and protein levels by analysis of variance with post-hoc Dunnett's t-tests. Error bars in figures indicate the S.E.

      DISCUSSION

      Our results indicate for the first time that the ELR+CXC chemokines LIX, KC, and MIP-2 up-regulate the proinflammatory cytokines IL-1β and TNF-α in cardiac-derived endothelial cells via activation of NF-κB. LIX-mediated NF-κB activation and κB-responsive gene transcription involves IκB hyperphosphorylation leading to its selective degradation in the cytoplasm by the proteasome system. The LIX signaling was inhibited by IL-10 and NF-κB pathway-specific mutant expression vectors. LIX signals via both CXCR1 and -R2 in inducing NF-κB activation. Specific blocking of CXCR2 attenuated NF-κB activation in rat cardiac-derived endothelial cells that express both R1 and R2, and completely abrogated LIX-induced NF-κB activation and κB-driven luciferase activity in mouse cardiac-derived endothelial cells that express only R2.
      The ELR+ CXC chemokines primarily attract and activate neutrophils to the site of injury/inflammation (
      • Oppenheim J.J.
      • Zachariae C.O.
      • Mukaida N.
      • Matsushima K.
      ,
      • Baggiolini M.
      • Dewald B.
      • Moser B.
      ,
      • Rollins B.J.
      ,
      • Schall T.J.
      • Bacon K.B.
      ,
      • Rovai L.E.
      • Herschman H.R.
      • Smith J.B.
      ,
      • Maekawa T.
      • Ishii T.
      ,
      • Simonini A.
      • Moscucci M.
      • Muller D.W.
      • Bates E.R.
      • Pagani F.D.
      • Burdick M.D.
      • Strieter R.M.
      ). We have previously shown that LIX, KC, and MIP-2 are expressed in the post-ischemic myocardium, and most notably LIX is expressed by all myocardial constituent cells (
      • Chandrasekar B.
      • Smith J.B.
      • Freeman G.L.
      ). Although activated neutrophils at the site of myocardial ischemic injury play a role in scavenging damaged tissue and subsequent remodeling, at least initially they exacerbate tissue injury through generation of free radicals, and secretion of various proteolytic enzymes and proinflammatory cytokines (
      • Jordan J.E.
      • Zhao Z.-Q.
      • Vinten-Johansen J.
      ,
      • Weiss S.J.
      ,
      • Engler R.L.
      • Schmid-Schonbein G.W.
      • Pavelec R.S.
      ,
      • Youker K.A.
      • Birdsall H.H.
      • Frangogiannis N.G.
      • Kumar A.G.
      • Lindsey M.L.
      • Ballantyne C.M.
      • Smith C.W.
      • Rossen R.D.
      • Entman M.L.
      ,
      • Frangogiannis N.G.
      • Youker K.A.
      • Entman M.L.
      ,
      • Williams F.M.
      ,
      • Boyle M.P.
      • Weisman H.F.
      ,
      • Entman M.L.
      • Smith C.W.
      ,
      • Entman M.L.
      • Youker K.
      • Shoji T.
      • Kukielka G.
      • Shappell S.B.
      • Taylor A.A.
      • Smith C.W.
      J..
      ). The results presented here demonstrate that chemokines (LIX) in addition to the recruitment of neutrophils to the site of myocardial ischemic injury may contribute to myocardial inflammation by the direct induction of cytokine expression.
      Interleukin-1β and TNF-α are κB-responsive proinflammatory cytokines with known negative myocardial inotropic effects. In isolated cardiomyocytes, papillary muscles, myocardial segments, Langendorff preparations, and in whole animals addition/infusion of TNF-α has been shown to depresses contractile function via induction of the inducible form of nitric-oxide synthase and sustained generation of high levels of nitric oxide (
      • Finkel M.S.
      • Oddis C.V.
      • Jacob T.D.
      • Watkins S.C.
      • Hattler B.J.
      • Simmons R.L.
      ,
      • Fridovich I.
      ,
      • Kelly R.A.
      • Smith T.W.
      ,
      • Le J.
      • Vilcek J.
      ,
      • Meldrum D.R.
      ,
      • Stylianou E.
      • Saklatvala J.
      ). High levels of TNF-α expression have also been shown to induce cell death by apoptosis (
      • Finkel M.S.
      • Oddis C.V.
      • Jacob T.D.
      • Watkins S.C.
      • Hattler B.J.
      • Simmons R.L.
      ). Both TNF-α and IL-1β are known free radical generators and NF-κB activators (
      • Bowie A.
      • O'Neill L.A.J.
      ). We have previously shown that TNF-α induces LIX via activation of NF-κB in isolated cardiomyocytes (
      • Chandrasekar B.
      • Smith J.B.
      • Freeman G.L.
      ). Furthermore, we demonstrated activation of NF-κB and induction of LIX by IL-1β and TNF-α in rat CDEC (Fig. 1). In the present study, we describe the converse, that is, the induction of proinflammatory cytokines by chemokines via NF-κB activation. Together, these observations indicate that NF-κB activation plays a central role in regulating cross-talk between chemokines and cytokines in myocardial cells.
      The ELR+ CXC chemokines bind and exert their biological effects via the seven-transmembrane heterotrimeric G protein-coupled receptors CXCR1 and CXCR2. The sequences of CXCR1 and -R2 within the seven-transmembrane domains and the connecting loops are homologous, but differ in the N and C-terminal domains, leading to overlapping as well as distinct ligand-binding and selective signal transduction pathways (
      • Ahuja S.K.
      • Murphy P.M.
      ,
      • Hall D.A.
      • Beresford I.J.
      • Browning C.
      • Giles H.
      ,
      • Jones S.A.
      • Wolf M.
      • Qin S.
      • Mackay C.R.
      • Baggiolini M.
      ,
      • Jones S.A.
      • Moser B.
      • Thelen M.
      ). Whereas all ELR+ CXC chemokines bind with high affinity to CXCR2, IL-8, because of the presence of Tyr13 and Lys15 in the N terminus has been shown to also bind R1 with high affinity (
      • Wolf M.
      • Delgado M.B.
      • Jones S.A.
      • Dewald B.
      • Clark-Lewis I.
      • Baggiolini M.
      ). Recently, granulocyte chemotactic protein-2 has also been shown to bind R1 with high affinity because of the presence of Arg20 (
      • Yang M.
      • Sang H.
      • Rahman A.
      • Wu D.
      • Malik A.B.
      • Ye R.D.
      ) indicating that other ELR+ CXC chemokines may bind to both R1 and R2 with high affinity. In the present study, we demonstrated that LIX-induced NF-κB activation is mediated in part through R2 in rat CDEC that express both R1 and R2, and fully through R2 in mouse CDEC that express only this receptor. Presumably, in the rat CDEC that express both receptors, the R2-independent signaling occurs through R1.
      Pretreatment of endothelial cells with pertussis toxin, which specifically blocks the coupling of CXC receptor to Giproteins, attenuated the LIX-induced increase in intracellular calcium levels and completely inhibited LIX-induced NF-κB activation. Similarly, treatment with LY 294002, a specific PI3K inhibitor, inhibited LIX-induced activation of PI3K activity and completely blocked NF-κB activation and κB-responsive cytokine gene transcription. In contrast, chelerythrine chloride, a PKC inhibitor, partially inhibited LIX-mediated NF-κB activation and cytokine expression. Collectively, these data indicate that LIX signals via inhibitory G proteins and PI3K, and partially via PKC. Although other G proteins may be involved in chemokine-mediated cell signaling (
      • Ye R.D.
      ,
      • Schraufstatter I.U.
      • Ma M.
      • Oades Z.G.
      • Barritt D.S.
      • Cochrane C.G.
      ), our results exclude this probability because LIX-mediated NF-κB activation was completely blocked by pertussis toxin.
      Interleukin-10 is an anti-inflammatory cytokine, and has been shown to block expression of various proinflammatory cytokines via inhibition of NF-κB activation (
      • Schottelius A.J.
      • Mayo M.W.
      • Sartor R.B.
      • Baldwin Jr., A.S.
      ). In the present study we demonstrate that IL-10 blocked LIX-induced NF-κB activation and κB-responsive gene transcription. It has been previously shown that IL-10 could block NF-κB activation by inhibiting IKK-mediated IκB phosphorylation and degradation (
      • Wolf M.
      • Delgado M.B.
      • Jones S.A.
      • Dewald B.
      • Clark-Lewis I.
      • Baggiolini M.
      ). Because the inhibitory effects of IL-10 are not cell-specific, and can inhibit activation of NF-κB in response to various proinflammatory stimuli, IL-10 may have a therapeutic potential in ischemia/reperfusion injury by blocking induction of proinflammatory cytokines and chemokines (
      • Frangogiannis N.G.
      • Youker K.A.
      • Rossen R.D.
      • Gwechenberger M.
      • Lindsey M.H.
      • Mendoza L.H.
      • Michael L.H.
      • Ballantyne C.M.
      • Smith C.W.
      • Entman M.L.
      ).
      In the present study, we demonstrate for the first time that inhibition of PARP-1 activation prevents LIX-mediated NF-κB activation and IL-1β and TNF-α expression. It has been demonstrated recently that PARP-1, a nuclear protein involved in repairing DNA strand breaks, has also been shown to activate NF-κB (
      • Ha H.C.
      • Hester L.D.
      • Snyder S.H.
      ). PARP-1 activation has been described during endotoxemia and inflammation (
      • Szabo C.
      • Lim L.H.
      • Cuzzocrea S.
      • Getting S.J.
      • Zingarelli B.
      • Flower R.J.
      • Salzman A.L.
      • Perretti M.
      ,
      • Oliver F.J.
      • Menissier-de Murcia J.
      • Nacci C.
      • Decker P.
      • Andriantsitohaina R.
      • Muller S.
      • de la Rubia G.
      • Stoclet J.C.
      • de Murcia G.
      ,
      • Kuhnle S.
      • Nicotera P.
      • Wendel A.
      • Leist M.
      ,
      • Liaudet L.
      • Pacher P.
      • Mabley J.G.
      • Virag L.
      • Soriano F.G.
      • Hasko G.
      • Szabo C.
      ,
      • Shall S.
      • de Murcia G.
      ). Administration of lipopolysaccharide to mice activated PARP-1 and resulted in PARP-1-dependent κB-responsive IL-1, IL-6, TNF-α, iNOS gene expression, and iNOS-mediated NO generation (
      • Oliver F.J.
      • Menissier-de Murcia J.
      • Nacci C.
      • Decker P.
      • Andriantsitohaina R.
      • Muller S.
      • de la Rubia G.
      • Stoclet J.C.
      • de Murcia G.
      ). Furthermore, in the murine system PARP-1 gene disruption or pharmacological inhibition of PARP-1 activation has been shown to reduce free radical generation, attenuate κB-responsive gene transcription, and reduce neutrophil infiltration in the lungs (
      • Kuhnle S.
      • Nicotera P.
      • Wendel A.
      • Leist M.
      ). Whether PARP-1 may be a logical target for inhibition to attenuate post-ischemic myocardial injury will require further study.
      Taken together, our results indicate that the ELR+ CXC chemokines, besides being potent neutrophil chemoattractants, also induce proinflammatory cytokine expression via activation of NF-κB. Blunting the activation of NF-κB or other components of the signaling cascade, rather than targeting inhibition of individual cytokines, chemokines, or adhesion molecules, may be a valid strategy to attenuate myocardial tissue injury during various inflammatory conditions.

      Acknowledgments

      We thank Doran W. Pearson for the nuclear run-on assay protocol (
      • Clark R.A.
      • Li S.L.
      • Pearson D.W.
      • Leidal K.G.
      • Clark J.R.
      • Denning G.M.
      • Reddick R.
      • Krause K.H.
      • Valente A.J.
      ), Lazaros C. Foukas for the PI3K assay protocol (
      • Foukas L.C.
      • Daniele N.
      • Ktori C.
      • Anderson K.E.
      • Jensen J.
      • Shepherd P.R.
      ), and Gregory L. Freeman for helpful discussions and critical review of the manuscript.

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