Nuclear Factor-κB Regulates Inflammatory Cell Apoptosis and Phagocytosis in Rat Carrageenin-Sponge Implant Model
2004; Elsevier BV; Volume: 165; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63280-4
ISSN1525-2191
AutoresMaria Chiara Maiuri, G Tajana, Teresa Iuvone, Daniela De Stefano, Guido Mele, M. Ribecco, Maria Pia Cinelli, Maria Fiammetta Romano, Maria Caterina Turco, Rosa Carnuccio,
Tópico(s)Bone Metabolism and Diseases
ResumoIn the present study we investigated whether apoptosis and phagocytosis are regulated by nuclear factor (NF)-κB in a model of chronic inflammation. The subcutaneous implant of λ-carrageenin-soaked sponges elicited an inflammatory response, characterized by a time-related increase of leukocyte infiltration into the sponge and tissue formation, which was inhibited by simultaneous injection of wild-type oligodeoxynucleotide decoy to NF-κB. Molecular and morphological analysis performed on infiltrated cells demonstrated: 1) an inhibition of NF-κB/DNA binding activity; 2) an increase of polymorphonuclear leukocyte apoptosis correlated either to an increase of p53 or Bax and decrease of Bcl-2 protein expression; and 3) an increase of phagocytosis of apoptotic polymorphonuclear leukocytes by macrophages associated with an increase of transforming growth factor-β1 and decrease of tumor necrosis factor-α as well as nitrite/nitrate production. Our results, showing that blockade of NF-κB by oligodeoxynucleotide decoy increases inflammatory cell apoptosis and phagocytosis, may contribute to lead to new insights into the mechanisms governing the inflammatory process. In the present study we investigated whether apoptosis and phagocytosis are regulated by nuclear factor (NF)-κB in a model of chronic inflammation. The subcutaneous implant of λ-carrageenin-soaked sponges elicited an inflammatory response, characterized by a time-related increase of leukocyte infiltration into the sponge and tissue formation, which was inhibited by simultaneous injection of wild-type oligodeoxynucleotide decoy to NF-κB. Molecular and morphological analysis performed on infiltrated cells demonstrated: 1) an inhibition of NF-κB/DNA binding activity; 2) an increase of polymorphonuclear leukocyte apoptosis correlated either to an increase of p53 or Bax and decrease of Bcl-2 protein expression; and 3) an increase of phagocytosis of apoptotic polymorphonuclear leukocytes by macrophages associated with an increase of transforming growth factor-β1 and decrease of tumor necrosis factor-α as well as nitrite/nitrate production. Our results, showing that blockade of NF-κB by oligodeoxynucleotide decoy increases inflammatory cell apoptosis and phagocytosis, may contribute to lead to new insights into the mechanisms governing the inflammatory process. The current paradigm indicates that the resolution of inflammation is an active process regulated by signals able to control leukocyte trafficking as well as gene expression that accompanies apoptosis and phagocytosis.1Savill J Apoptosis in resolution of inflammation.J Leukoc Biol. 1997; 61: 375-380Crossref PubMed Scopus (561) Google Scholar, 2Kobayashi SD Voyich JM Buhl CL Stahl RM DeLeo FR Global changes in gene expression by human polymorphonuclear leukocytes during receptor-mediated phagocytosis: cell fate is regulated at the level of gene expression.Proc Natl Acad Sci USA. 2002; 99: 6901-6906Crossref PubMed Scopus (198) Google Scholar, 3Kobayashi SD Voyich JM Somerville JA Braughton KR Malech HL Musser JM DeLeo FR An apoptosis-differentiation program in human polymorphonuclear leukocytes facilitates resolution of inflammation.J Leukoc Biol. 2003; 73: 315-322Crossref PubMed Scopus (64) Google Scholar The transition from acute to chronic inflammation occurs when these signals might be absent or become dysregulated, although the mechanisms underlying the development of chronic inflammation are primarily undefined. In the last years numerous studies in vitro have shown that apoptosis and phagocytosis play a key role in promoting the resolution of inflammation.4Savill J Dransfield I Gregory C Haslett C A blast from the past: clearance of apoptotic cells regulates immune responses.Nat Rev. 2002; 2: 965-975Crossref Google Scholar Polymorphonuclear leukocytes (PMNs), the first cells infiltrating into the inflamed site, are constitutively programmed to undergo apoptosis, subsequently recognized and ingested by neighboring macrophages.5Savill J Wyllie AH Henson JE Walport MJ Henson PM Haslett C Macrophage phagocytosis of aging neutrophils in inflammation. 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Indeed, disorder of apoptosis leading to leukocyte longevity as well as defective clearance of apoptotic cells has been suggested to contribute to the development of chronic inflammation.14Stern M Savill J Haslett C Human monocyte-derived macrophage phagocytosis of senescent eosinophils undergoing apoptosis. 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For this purpose, we used synthetic double-stranded oligodeoxynucleotide (ODN) decoy to NF-κB capable of blocking the transcriptional activity of NF-κB.36Morishita R Sugimoto T Aoki M Kida I Tomita N Moriguchi A Maeda K Sawa Y Kaneda Y Higaki J Ogihara T In vivo transfection of cis element “decoy” against nuclear factor-kappaB binding site prevents myocardial infarction.Nat Med. 1997; 3: 894-899Crossref PubMed Scopus (604) Google Scholar, 37Miagkov AV Kovalenko DV Brown CE Didsbury JR Cogswell JP Stimpson SA Baldwin AS Makarov SS NF-kappaB activation provides the potential link between inflammation and hyperplasia in the arthritic joint.Proc Natl Acad Sci USA. 1998; 95: 13859-13864Crossref PubMed Scopus (413) Google Scholar, 38Tomita T Takeuchi E Tomita N Morishita R Kaneko M Yamamoto K Nakase T Seki H Kato K Kaneda Y Ochi T Suppressed severity of collagen-induced arthritis by in vivo transfection of nuclear factor-kappaB decoy oligodeoxynucleotides as a gene therapy.Arthritis Rheum. 1999; 42: 2532-2542Crossref PubMed Scopus (222) Google Scholar, 39D'Acquisto F Ialenti A Ianaro A Di Vaio R Carnuccio R Local administration of transcription factor decoy oligonucleotides to nuclear factor-kappaB prevents carrageenin-induced inflammation in rat hind paw.Gene Ther. 2000; 7: 1731-1737Crossref PubMed Scopus (53) Google ScholarMaterials and MethodsAnimalsMale Wistar rats (Harlan, Udin, Italy), weighing 180 to 250 g, were used in all experiments. Animals were provided with food and water ad libitum. The light cycle was automatically controlled (on 7 hours; off 19 hours) and the room temperature thermostatically regulated to 22 ± 1°C with 60 ± 5% humidity. Before the experiments, animals were housed in these conditions for 3 to 4 days to become acclimatized. Animal care was in accordance with the Italian and European regulations on protection of animals used for experimental and other scientific purposes.Sponge Implant ModelSponge implant in the rat was performed as previously described.40Iuvone T Carnuccio R Di Rosa M Modulation of granuloma formation by endogenous nitric oxide.Eur J Pharmacol. 1994; 265: 89-92Crossref PubMed Scopus (57) Google Scholar Briefly, two polyether cylindrical sponges (190 mm in length, 10 mm in diameter) for the same treatment were implanted subcutaneously on the back of rats under general anesthesia. Sponges and surgery tools were autoclaved. λ-Carrageenin (1% w/v) was prepared in sterile pyrogen-free saline and endotoxin contamination was evaluated by the Limulus test. After implant, 0.5 ml of 1% λ-carrageenin was injected into each sponge in the presence or absence of wild-type ODN (W.T., 20 μg/site) or mutant ODN (Mut, 20 μg/site) decoy using saline as vehicle. After sponge implant (1, 3, and 5 days) rats were sacrificed in an atmosphere of CO2. The granulomatous tissue around the sponge was dissected and weighted.Collection of Cells and ExudateThe sponges were removed and centrifuged at 400 × g for 15 minutes. The exudate volume recovered from the sponge was measured and stored at −20°C. The cell pellet was suspended in 1 ml of saline and total cell amount was counted by phase-contrast microscopy. In some experiments total cells (1 × 106) were resuspended in 100 μl of 5% bovine serum albumin/phosphate-buffered saline (PBS) and incubated with 10 μl of fluorescein isothiocyanate-conjugated anti-granulocyte antibody (BD Pharmingen, San Diego, CA) for 15 minutes in the dark at room temperature. The cells were washed and resuspended in 500 μl of PBS and then analyzed by a FACScan 30 (BD) flow cytometer. A threshold was set for FSc to exclude the debris and white blood cell populations were identified using a SSc/Fl-1 or SSc/FSc dual-parameter plot. Cytosolic and nuclear extracts of cells collected from sponges were prepared as previously described.28D'Acquisto F De Cristofaro F Maiuri MC Tajana G Carnuccio R Protective role of nuclear factor kappaB against nitric-oxide apoptosis in J774 macrophages.Cell Death Differ. 2001; 8: 144-151Crossref PubMed Scopus (27) Google Scholar Protein concentration was determined by Bio-Rad (Milan, Italy) protein assay kit.TGF-β1, Tumor Necrosis Factor (TNF)-α, and Nitrite/Nitrate (NOx) DeterminationThe accumulation of TGF-β1 and TNF-α in the inflammatory exudate was measured by an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions (TGF-β1, Emax ImmunoAssay System; Promega, Madison, WI; TEMA Ricerche, Milan, Italy). The amount of NOx, stable metabolites of nitric oxide, was determined as previously described.41D'Acquisto F Ialenti A Ianaro A Carnuccio R Nuclear factor-κB activation mediates inducile nitric oxide synthase expression in carrageenin-induced rat pleurisy.N-S Arch Pharmacol. 1999; 360: 670-675Crossref PubMed Scopus (12) Google ScholarHistological and Immunohistochemical InvestigationsExplants were fixed in formol-methanol (9:1, v/v) solution at 4°C for 24 hours. After dehydration in an ethanol series and infiltration with xylol, paraffin wax sections were cut at 4 to 6 μm and stained with hematoxylin and eosin or Mallory's triple stain. The cellular migration into the sponge was evaluated by counting 100 cells per well at ×1000 magnification under oil immersion. Immunostaining of sections was performed for p65, p53, Bax, and Bcl-2 antibodies using a streptavidin-biotin immunoperoxidase technique. Chromogen reaction was developed with diaminobenzidine solution (DAKO, Milan, Italy). Phagocytosis was evaluated by counting 200 to 300 macrophages per well at ×1000 magnification under oil immersion. Results were expressed as percentage of monocyte-derived macrophages (MΦ) containing at least one ingested apoptotic PMN (phagocytic percent).Ultrastructure InvestigationsImmediately after explant the granuloma was rinsed in phosphate-buffered saline (PBS), then immersed in a mixture of 2% glutaraldehyde and 1% formaldehyde in 0.1 mol/L of phosphate buffer, pH 7.2, for 2 hours. The specimens were divided and used for investigations by scanning electron microscopy and transmission electron microscopy.Scanning Electron MicroscopyAfter primary fixation the specimens were dehydrated in acetone and dried in a critical point drier CPD010 (Balzers Union, Liechtenstein) with carbon dioxide. The specimens were mounted on the aluminum mounts and sputtered in Coating Unit E5100 (Polaron, UK) with gold-palladium and examined under a Tesla BS340 microscope. The pictures were taken and additionally enlarged to the desired magnification at ×3000.Transmission Electron MicroscopyDuring the primary fixation the samples were divided into ∼1- to 2-mm3 blocks for the better fixation. The primary fixation was followed by a 2-hour secondary fixation in 2% osmium tetraoxide in the 0.1 mol/L phosphate buffer. After dehydration in acetone with uranium acetate and embedding in Durcupan ACM (Fluka, Milan, Italy), semi-thin toluidine blue-stained sections were made for the control in the light microscope. From the selected areas target ultra-thin section were cut, stained by uranium acetate and lead citrate, and examined and photographed under the transmission electron microscope (Opton 109, Zeiss, Milan, Italy) at the accelerating voltage 80 kV. The pictures were taken at the primary magnification of ×1000 to ×50,000 and additionally enlarged to desired magnification.Apoptosis AnalysisPMN apoptosis was evaluated through morphological criteria by light microscopy. Criteria used to diagnose apoptosis included chromatin aggregation, cytoplasmic vacuolation, and/or cell shrinkage.5Savill J Wyllie AH Henson JE Walport MJ Henson PM Haslett C Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages.J Clin Invest. 1989; 83: 865-875Crossref PubMed Scopus (1336) Google Scholar Moreover, analysis of apoptosis was performed by flow cytometry using annexin V that binds to phosphatidylserine exposed on the surface of apoptotic cells. To measure granulocyte apoptosis, 1 × 106 cells were incubated, in double staining, with annexin V phycoerythrin-conjugated (Alexis, San Diego, CA) plus the fluorescein isothiocyanate-anti-rat granulocyte monoclonal antibody (Pharmingen/Becton Dickinson, San Diego, CA) in 100 μl of binding buffer containing 10 mmol/L Hepes/NaOH, pH 7.5, 140 mmol/L NaCl, and 2.5 mmol/L CaCl2 for 15 minutes at room temperature in the dark. Then 400 μl of the same buffer were added to each sample and the cells were analyzed by a Becton Dickinson FACScan flow cytometer. The percentage of annexin-positive cells was referred to the gated (FL1/SSC) granulocyte.Transcription Factor Decoy OligonucleotidesPlain double-stranded ODN decoys to NF-κB were prepared by annealing of sense and anti-sense phosphorothioate oligonucleotides in vitro in 1× annealing buffer (20 mmol/L Tris-HCl, pH 7.5, 20 mmol/L MgCl2, and 50 mmol/L NaCl). The mixture was heated at 100°C for 12 minutes and allowed to cool to room temperature slowly throughout 18 hours.The sequence of ODN decoy to NF-κB (W.T. ODN) used was: wild-type NF-κB consensus sequence 5′-GAT CGA GGG GAC TTT CCC TAG C-3′ and 3′-CTA GCT CCC CTG AAA GGG ATC G-5′; mutant NF-κB consensus sequence with a mutation of the bolded bases (GGAC to AAGC) of wild-type NF-κB consensus sequence (Mut ODN) 5′-GAT CGA GGA AGC TTT CCC TAG C-3′ and 3′-CTA GCT CCT TCG AAA GGG ATC G-5′.Electrophoretic Mobility Shift AssayDouble-stranded oligonucleotides containing the NF-κB recognition sequence (5′-CAACGGCAGGGGAATCTCCCTCTCCTT-3′) as well as W.T. ODN were end-labeled with 32P-γ-ATP. Nuclear extracts containing 5 μg of protein were incubated for 15 minutes with radiolabeled oligonucleotides (2.5 to 5.0 × 104 cpm) in a reaction buffer as previously described.28D'Acquisto F De Cristofaro F Maiuri MC Tajana G Carnuccio R Protective role of nuclear factor kappaB against nitric-oxide apoptosis in J774 macrophages.Cell Death Differ. 2001; 8: 144-151Crossref PubMed Scopus (27) Google Scholar The specificity of the NF-κB/DNA binding was determined by a competition reaction in which a 50-fold molar excess of unlabeled wild-type, mutant, or Sp-1 oligonucleotide was added to the binding reaction 15 minutes before the radiolabeled probe. The specificity of the ODN/DNA binding was determined by competition reaction in which a 50-fold molar excess of unlabeled wild-type or mutant ODN was added to the binding reaction 15 minutes before the radiolabeled probe. In the supershift assay, antibodies reactive to p52, c-Rel, RelB, p50, or p65 proteins were added to the reaction mixture 15 minutes before the addition of the radiolabeled NF-κB probe. Nuclear protein-oligonucleotide complexes were resolved by electrophoresis on a 6% nondenaturing polyacrylamide gel in 1× TBE (Tris borate-ethylenediaminetetraacetic acid) buffer at 150 V for 2 hours at 4°C. The gel was dried and autoradiographed with intensifying screen at −80°C for 20 hours. Subsequently, the relative bands were quantified by densitometric scanning of the X-ray films with a GS-700 Imaging Densitometer (Bio-Rad) and a computer program (Molecular Analyst; IBM).Western Blot AnalysisImmunoblotting analysis of anti-Bax, anti-Bcl-2, anti-p53, anti-histone 1, anti-p50, and anti-p65 was performed on cytosolic or nuclear fraction, respectively, as previously described.28D'Acquisto F De Cristofaro F Maiuri MC Tajana G Carnuccio R Protective role of nuclear factor kappaB against nitric-oxide apoptosis in J774 macrophages.Cell Death Differ. 2001; 8: 144-151Crossref PubMed Scopus (27) Google Scholar Cytosolic and nuclear fraction proteins were mixed with gel loading buffer in a ratio of 1:1, boiled, and centrifuged at 10,000 × g. Protein concentration was determined and equivalent amounts (50 μg) of each sample were electrophoresed in a 8% discontinuous polyacrylamide minigel. The proteins were transferred onto nitro-cellulose membranes, according to the manufacturer's instructions (Bio-Rad). The membranes were saturated by incubation at room temperature for 2 hours with 10% nonfat dry milk in PBS and then incubated with (1:1000) anti-Bax, anti-Bcl-2, anti-p53, anti-p50, and anti-p65 at 4°C overnight. The membranes were washed with 0.1% Tween 20 in PBS and then incubated with anti-rabbit, anti-mouse, or anti-goat immunoglobulins coupled to peroxidase (1:1000) (DAKO, Milan, Italy). The immunocomplexes were visualized by the enhanced chemiluminescence method (Amersham, Milan, Italy). The membranes were stripped and reprobed with β-actin antibody to verify equal loading of proteins. Subsequently, the relative expression of Bax, Bcl-2, p53, p50, and p65 in cytosolic and nuclear fraction was quantified by densitometric scanning of the X-ray films with a GS 700 Imaging Densitometer (Bio-Rad) and a computer program (Molecular Analyst, IBM).StatisticsStatistical significance was calculated by one-way analysis of variance and Bonferroni-corrected P value for multiple comparison test. The level of statistically significant difference was defined as P < 0.05. The fold increase was calculated by dividing the combination value by sum of individual values.ReagentsOligonucleotide synthesis was performed to our specifications by Tib Molbiol, Boehringer-Mannheim (Genova, Italy). 32P-γ-ATP was from Amersham (Milan, Italy). Poly dI-dC was from Boehringer-Mannheim (Milan, Italy). Anti-p50, anti-p65, anti-p52, anti-c-Rel, anti-RelB, and anti-histone 1 antibodies were from Santa Cruz (Milan, Italy). Anti-Bax, anti-Bcl-2, and anti-β-actin antibodies were from Oncogene (Milan, Italy). Anti-p53 antibody was from Pharmingen/Becton Dickinson (San Diego, CA). PBS and Tween 20 were from ICN (Milan, Italy). Nonfat dry milk was from Applichem (Darmstadt, Germany). All other reagents were from Sigma (Milan, Italy).ResultsODN Decoy to NF-κB Reduces Infiltrating Leukocytes and Granulomatous Tissue Formationλ-Carrageenin (1%) induced a significant increase in infiltrating leukocytes and granulomatous tissue formation after 1, 3, and 5 day (versus saline). The exudate volume did not change throughout the time and treatment. The total number of infiltrated leukocytes into the sponge was highest at day 1, and then declined time dependently. Conversely, tissue weight was further increased up to day 5. Local injection of W.T. ODN decoy to NF-κB (20 μg/site), but not Mut ODN decoy (20 μg/site), inhibited leukocyte infiltration (by 51.65 ± 0.62%, 59.34 ± 0.45%, and 57.36 ± 0.4%, P < 0.0001; n = 15 to 20 sponges from 9 to 10 rats) and granuloma formation (by 38.96 ± 0.06%, 45.0 ± 0.04%, and 38.03 ± 0.09%, P < 0.0001; n = 15 to 20 sponges from 9 to 10 rats) as compared to carrageenin alone at time points considered (Figure 1, A and B). The migration and concomitant tridimensional organization of leukocytes into the sponge was studied by light microscopy and scanning electron microscopy, respectively (Figure 2, A and B). A first identification of cell activation status as well as apoptosis and phagocytosis was performed by transmission electron microscopy (Figure 2C). We observed that W.T. ODN decoy treatment caused a reduced migration accompanied with changed arrangement of cell populations in the sponge. Particularly, transmission electron microscopy analysis indicated a marked leukocyte activation associated with pictures of apoptosis and phagocytosis. The inflammatory cell profile, determined by light microscopy, is illustrated in Figure 3. The PMN contribution to the infiltrate was maximal on day 1 and declined after 3 days, whereas lymphocyte/monocyte/macrophage infiltration increased progressively up to 5 days. The W.T. ODN decoy did not affect the cellular profile. However, flow cytometry analysis confirmed the composition of the cells infiltrated into the sponge using both SSc/FL-1 (fluorescein isothiocyanate-conjugated anti-granulocyte antibody) and SSc/FSc dual-parameter plots. The profile of leukocytes from the λ-carrageenin-treated sponge was the following: 90% granulocytes, 4% lymphocytes, and 6% monocytes/macrophages on day 1; 64% granulocytes, 15% lymphocytes, and 21% monocytes/macrophages on day 3. These results were comparable to those obtained from the saline-, W.T. ODN-, and Mut ODN-treated sponges. Unfortunately, we were not able to analyze the samples on day 5 because the cells apparently have lost the typical FSc and SSc patterns.Figure 2Morphological summary depicting cell migration, tridimensional organization, and activation status in the sponge after treatments. A: Light microscopy: relationship between spaces invaded by cells (outlined areas) and exudate as well as debris. B: Scanning electron microscopy: presence of cells organized even in clusters with debris (black asterisk) and exudate (white asterisk) areas. C: Transmission electron microscopy: ultrastructural features of cell populations with cytosol suggestive of phagocytosis as well as autophagy (open arrows) and nuclei involution (filled arrows) Fields are representative o
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