Naunyn-Schmiedeberg’s Arch Pharmacol (2001) 364 : 422–429 DOI 10.1007/s002100100472
O R I G I N A L A RT I C L E
Fulvio D’Acquisto · Angela Ianaro · Armando Ialenti · Pasquale Maffia · Maria Chiara Maiuri · Rosa Carnuccio
Transcription factor decoy oligodeoxynucleotides to nuclear factor-κB inhibit reverse passive Arthus reaction in rat Received: 4 May 2001 / Accepted: 13 July 2001 / Published online: 4 September 2001 © Springer-Verlag 2001
Abstract In the present study we investigated in the reverse passive Arthus reaction elicited in the rat skin the anti-inflammatory effect of double-stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-κB (NF-κB) sequence as transcription factor decoys (TFD) to inhibit NF-κB binding to native DNA sites. Local administration of wild-type-, but not mutant-decoy ODN, dose-dependently reduced both plasma leakage and neutrophil infiltration in rat skin. Molecular analysis performed on soft tissue obtained from rat skin demonstrated: (1) an inhibition of NF-κB/DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-κB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-κB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-κB complexes, as well as ammonium pyrrolidine dithiocarbamate, an inhibitor of NF-κB activation, exhibited a similar profile of activity of decoy ODN. Our results indicate that decoy ODN, acting as an in vivo competitor for the transcription factor’s ability to bind to cognate recognition sequence, may represent a novel strategy to modulate immune reactions. Keywords Cyclooxygenase-2 · Inducible nitric oxide synthase · Nuclear factor-κB · Reverse passive Arthus reaction · Transcription factor decoy
Introduction The reverse passive Arthus reaction (RPA) is a model of immune complex-mediated microvascular injury and type
F. D’Acquisto · A. Ianaro · A. Ialenti · P. Maffia · M.C. Maiuri · R. Carnuccio (✉) Department of Experimental Pharmacology, University of Naples “Federico II”, Via Domenico Montesano, 49, 80131 Naples, Italy e-mail:
[email protected], Fax: +39-81-678403
III hypersensitivity reaction which can be elicited in the skin of laboratory animals by the intradermal injection of an antibody followed by an intravenous injection of its cognate antigen. The sequence of events that occur in the reverse passive Arthus reaction includes formation of immune complexes in the microvessel wall, activation of the complement cascade, migration and adhesion of polymorphonuclear leukocytes to the endothelial cells, platelet accumulation, oedema and haemorrhage (Crawford et al. 1982). Immune complexes initiate the generation of various inflammatory mediators including nitric oxide (NO) and prostaglandins (PGs) which can influence the accumulation and activation state of inflammatory cells (Mulligan et al. 1991; Moreno 1993). Some studies have shown the involvement of NO in the production of tissue injury in a rat model of alveolitis and dermal vasculitis similar to RPA (Mulligan et al. 1991, 1992). We have recently shown that NO plays a relevant role as a modulator of the RPA reaction elicited in rat skin (Ianaro et al. 1998). It is well known that PGs are important mediators of inflammation (Di Rosa et al. 1971) and it has been shown that intradermally injection of PGs in guinea-pig skin or in rabbit skin enhances local oedema formation and neutrophil accumulation (Williams and Peck 1977; Teixera et al. 1993). It has been demonstrated that both NO and PGs released at inflammatory site are generated by the inducible isoforms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2), respectively (Ialenti et al. 1992; Seibert et al. 1994). The promoter region of both COX-2 (Sirois et al. 1993) and iNOS genes (De Vera et al. 1996) has been cloned and sequenced. These promoter regions contain at least one putative NF-κB consensus sequence that has been shown to act as positive regulatory element for both COX-2 and iNOS transcription (Xie et al. 1994; Yamamoto et al. 1995). NF-κB is a member of the Rel family proteins and is typically a heterodimer of p50 and p65 subunit (Baeuerle and Baltimore 1996). Each member of this family contains a conserved N-terminal region called the Rel-homology domain (RHD) within which lie the DNA-binding and dimerization domains and the nuclear localization sequences (NLS). In quiescent cells, NF-κB resides in the cytosol in latent form
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bound to inhibitory proteins, IκBs. Stimulation of different types of cells with lipopolysaccharide, cytokines or oxidants triggers a series of signalling events that ultimately converge to the activation of one or more redoxsensitive kinases which specifically phosphorylate IκB, resulting in IκB polyubiquinitation and subsequent degradation (Baeuerle and Henkel 1994). Once activated, the liberated NF-κB translocates into the nucleus and stimulates transcription by binding to cognate κB sites in the promoter regions of various target genes (May and Ghosh 1998). We have recently shown that NF-κB is activated in the carrageenin-induced rat pleurisy and its inhibition by ammonium pyrrolidine dithiocarbamate, an antioxidant inhibitor of NF-κB (Schreck et al. 1992), was associated with a reduction of both exudate formation and leukocyte infiltration (D’Acquisto et al. 1999a, 1999b). Several studies on the activation pathway of NF-κB led to the discovery of new molecules capable of blocking the transcriptional activity of this transcription factor (Pierce et al. 1997). Thus, recent evidences have demonstrated that synthetic double-stranded oligodeoxynucleotides (ODN) as decoy cis-elements block the binding of NF-κB to promoter regions of its targeted genes, resulting in the inhibition of gene transactivation both in vitro (Sharma et al. 1996; Schmedtje et al. 1997) and in vivo (D’Acquisto et al. 2000). An alternative approach utilizes a cell-permeable peptide, called SN-50, carrying a functional domain NLS capable of inhibiting the nuclear translocation of NF-κB complexes (Lin et al. 1995). In the present study we investigated the anti-inflammatory effect of double-stranded decoy ODN NF-κB on a model of type III hypersensitivity such as rat dermal RPA reaction. We provide evidence that ODN decoy to NF-κB inhibits inflammatory reaction, evaluated as plasma leakage and neutrophil infiltration at the challenged sites, and reduces both COX-2 and iNOS protein expression.
Materials and methods Animals. Male Wistar rats (Harlan, Italy), weighing 200–250 g, were used in all experiments. Animals were provided with food and water ad libitum. The light cycle was automatically controlled (on 07 h 00 min; off 19 h 00 min) and the room temperature thermostatically regulated to 22±1°C. Prior to the experiments, animals were housed in these conditions for 3–4 days to become acclimatized. Animal care was in accordance with Italian and European regulations on protection of animals used for experimental and other scientific purposes. Transcription factor decoy oligonucleotides. Plain double-stranded decoy ODN to NF-κB were prepared by annealing of sense and antisense oligonucleotides in vitro in 1× annealing buffer (20 mM Tris-HCl, pH 7.5, 20 mM MgCl2 and 50 mM NaCl). The mixture was heated at 100°C for 12 min and allowed to cool to room temperature slowly over 18 h. The sequence of decoy ODN to NF-κB used was: 1. wild-type-NF-κB consensus sequence 5′-GAT CGA GGG GAC TTT CCC TAG C-3′ 3′-CTA GCT CCC CTG AAA GGG ATC G-5′ 2. mutant-NF-κB consensus sequence with a mutation of the bolded bases (GGAC to AAGC) of wild-type NF-κB consensus sequence
5′-GAT CGA GGA AGC TTT CCC TAG C-3′ 3′-CTA GCT CCT TCG AAA GGG ATC G-5′. Induction of RPA reaction. The reverse passive Arthus reaction was induced using bovine serum albumin as antigen and rabbit antibovine serum albumin as antibody. Rats were lightly anaesthetized with isoflurane and the hair was shaved from the mid-dorsal region. After shaving each animal was injected intradermally (i.d.) into six skin sites, according to a balanced pattern, with 0.1 ml pyrogen-free phosphate buffer saline (PBS) containing 25 µg of antibovine serum albumin in the absence or presence of test agents mixed with the anti-bovine serum albumin preparations immediately prior to i.d. injection. The agents were as follows: wild typedecoy ODN (3–10–30 µg/site), mutant-decoy ODN (30 µg/site), SN-50 (10 µg/site), mutant SN-50 (10 µg/site) and ammonium pyrrolidine dithiocarbamate (PDTC, 1–3–10 µg/site). Immediately following the i.d. injection, each rat received 0.5 ml of PBS containing 1 mg bovine serum albumin injected in the tail vein. The animals were re-anaesthetized with isoflurane 6 h after challenge and were then sacrificed by cervical dislocation. The full thickness skin was removed from the back of each animal and a disc 10 mm in diameter was punched out with a metal punch. The samples were then processed for the evaluation of vascular permeability and neutrophil infiltration as well as to obtain cytosolic and nuclear extracts (see below). Vascular permeability. Local increase in vascular permeability was measured as plasma leakage at skin sites in response to RPA reaction. Evans blue (2% in pyrogen-free PBS) was injected via tail vein (30 mg/kg) 5 min after the induction of the RPA reaction. The extraction of Evans blue from the skin samples was performed as described by Humphrey (1993) and the amount of Evans blue in the samples was calculated from a standard curve. Plasma leakage was expressed as µg of Evans blue in the skin samples. Neutrophil infiltration. Myeloperoxidase, a haemoprotein located in azurophil granules of neutrophils, has been used as an enzyme marker of neutrophil infiltration in various tissues (Bradley et al. 1982). Myeloperoxidase extraction from each skin sample was performed according to the procedure described by Bailey and Sturm (1983). Myeloperoxidase activity was measured by the change in optical density (at 650 nm) resulting from decomposition of H2O2 in the presence of 3,3′,5,5′-tetramethylbenzidine as a hydrogen donor (Schierwagen et al. 1990). It was expressed as the number of rat peritoneal neutrophils (glycogen elicited and purified as described by McCall et al. 1989) containing an equivalent amount of myeloperoxidase. Cytosolic and nuclear extracts. In a separate set of experiments rat skin sites removed 6 h after RPA induction were immediately and separately processed to obtained cytosolic and nuclear extracts as previously described with some modification (D’Acquisto et al. 1999c). Briefly, each inflamed skin site was frozen in liquid nitrogen, immediately suspended in 6 ml of ice-cold hypotonic lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenylmethylsulphonylfluoride, 1.5 µg/ml soybean trypsin inhibitor, 7 µg/ml pepstatin A, 5 µg/ml leupeptin, 0.1 mM benzamidine, 0.5 mM dithiothreitol) and homogenised at the highest setting for 2 min in a Polytron PT 3000 (AG Kinematica) tissue homogenizer. The homogenates were divided in three aliquots of 2 ml, chilled on ice for 15 min and then vigorously shaken for another 15 min in the presence of 20 µl of 10% Nonidet P-40. The nuclear fraction was precipitated by centrifugation at 1500 g for 5 min, the supernatant containing the cytosolic fraction was removed and stored at –80°C. The nuclear pellet was resuspended in 700 µl of high salt extraction buffer (20 mM pH 7.9 HEPES, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% v/v glycerol, 0.5 mM phenylmethylsulphonylfluoride, 1.5 µg/ml soybean trypsin inhibitor, 7 µg/ml pepstatin A, 5 µg/ml leupeptin, 0.1 mM benzamidine, 0.5 mM dithiothreitol) and incubated with shaking at 4°C for 30 min. The nuclear extract was then centrifuged for 15 min at 13,000 g and supernatant was aliquoted and stored at –80°C. Protein concentration was determined by Bio-Rad (Milan, Italy) protein assay kit.
424 Electrophoretic mobility shift assay. A double-stranded oligonucleotide containing the NF-κB recognition sequence (5′-CCA ACT GGG GAC TCT CCC TTT G-3′) was end-labeled with [32P]γ-ATP. Nuclear extracts (40 µg) from each skin site were incubated for 30 min with radiolabeled oligonucleotides (2.5–5.0×104 cpm) in 20 µl reaction buffer containing 2 µg poly dI-dC, 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mg/ml bovine serum albumin, 10% (v/v) glycerol. The specificity of the DNA/protein binding was determined by competition reaction in which a 50-fold molar excess of unlabeled wild-type, mutant or Sp-1 oligonucleotide was added to the binding reaction 10 min before addition of radiolabeled probe. In supershift assay, antibodies reactive to p50, p65 or c-Rel proteins were added to the reaction mixture 30 min before the addition of radiolabeled NF-κB probe. Nuclear protein-oligonucleotide complexes were resolved by electrophoresis on a 6% non-denaturing polyacrylamide gel in 1× Tris borate EDTA buffer at 150 V for 2 h at 4°C. The gel was dried and autoradiographed with an intensifying screen at –80°C for 20 h. Subsequently, the relative bands were quantified by densitometric scanning of the X-ray films with a GS-700 Imaging Densitometer (Bio-Rad, Milan, Italy) and a computer program (Molecular Analyst; IBM). Immunoprecipitation and Western blot analysis. The level of p50 and p65 and the expression of COX-2 and iNOS were quantified in nuclear and cytosolic extracts, respectively, by immunoprecipitation followed by Western blot analysis according to the manufacturer’s instructions (Santa Cruz, Milan, Italy). Briefly, protein concentration was determined and equivalent amounts (200 µg) for each sample were mixed with 40 µl of protein A-sepharose and 2 µl of anti-p50, anti-p65, anti-COX-2 or anti-iNOS polyclonal antibodies and left overnight at 4°C with continuous shaking. Immunocomplexes were washed three times with 500 µl of buffer A (10 mM Tris-HCl pH 7.5, 1 M NaCl, 0.2% Triton-X 100 and 2 mM EDTA), mixed with 40 µl of gel loading buffer (50 mM Tris/10% SDS/10% glycerol/10% 2-mercaptoethanol/2 mg of bromophenol per ml) and then boiled for 3 min. Samples so obtained were electrophoresed in a 12% discontinuous polyacrylamide minigel. The proteins were transferred onto nitrocellulose membranes, according to the manufacturer’s instructions (Bio-Rad). The membranes were saturated by incubation at 4°C overnight with 10% non-fat dry milk in PBS and then incubated with anti-p50, anti-p65, antiCOX-2 or anti-iNOS antibodies for 1 h at room temperature. The membranes were washed three times with 1% Triton-X 100 in PBS and then incubated with anti-rabbit or anti-goat immunoglobulins coupled to peroxidase. The immunocomplexes were visualized by the ECL chemiluminescence method (Amersham, Milan, Italy). Subsequently, the relative bands were quantified by densitometric scanning of the X-ray films with a GS-700 Imaging Densitometer (Bio-Rad, Milan, Italy) and a computer program (Molecular Analyst; IBM). β-Actin (Sigma, Milan, Italy) Western blot analysis was performed to ensure equal sample loading.
Results Effect of PDTC, decoy ODN and SN-50 on RPA reaction The induction of the RPA reaction in the rat skin (positive control) elicited an inflammatory reaction characterized by both local increase in vascular permeability and neutrophil infiltration at the challenged skin site with a peak response occurring at 6 h. As shown in Fig. 1A,B, in positive controls plasma leakage, evaluated as Evans blue extravasation, was 26.6±0.5 µg/site (n=16), while neutrophil recruitment, evaluated as myeloperoxidase activity, was equivalent to 27.1±0.6×104 neutrophil equivalents/site (n=16). The omission of i.v. bovine serum albumin injection (negative control) produced an almost undetectable Evans blue extravasation (1.9±0.2 µg/site, n=6) and myeloperoxidase activity (0.020±0.001×104 neutrophil equivalents/site, n=6). In preliminary experiments we established that neither the Evans blue extravasation nor the myeloperoxi-
Statistics. Statistical significance was calculated by one-way analysis of variance (ANOVA) and Bonferroni-corrected P-value for multiple comparison test. Triplicate skin responses were averaged and the mean ± SEM of these responses in n animals was calculated. The level of statistically significant difference was defined as P
