Inhibition of Macrophage Nuclear Factor-κB Leads to a Dominant Anti-Inflammatory Phenotype that Attenuates Glomerular Inflammation in Vivo
2005; Elsevier BV; Volume: 167; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)62950-1
ISSN1525-2191
AutoresHeather M. Wilson, Salah Chettibi, Christian Jobin, David Walbaum, Andrew J. Rees, David Kluth,
Tópico(s)Inflammatory mediators and NSAID effects
ResumoInfiltrating macrophages (mϕ) can cause injury or facilitate repair, depending on how they are activated by the microenvironment. Studies in vitro have defined the roles of individual cytokines and signaling pathways in activation, but little is known about how macrophages integrate the multiple signals they receive in vivo. We inhibited nuclear factor-κB in bone marrow-derived macrophages (BMDMs) by using a recombinant adenovirus expressing dominant-negative IκB (Ad-IκB). This re-orientated macrophage activation so they became profoundly anti-inflammatory in settings where they would normally be classically activated. In vitro, the lipopolysaccharide-induced nitric oxide, interleukin-12, and tumor necrosis factor-α synthesis was abrogated while interleukin-10 synthesis increased. In vivo, fluorescently labeled BMDMs transduced with Ad-IκB and injected into the renal artery significantly reduced inducible nitric oxide synthase and MHC class II expression when activated naturally in glomeruli of rats with nephrotoxic nephritis. Furthermore, although they only comprised 15% of glomerular macrophages, their presence significantly reduced glomerular infiltration and activation of host macrophages. Injury in nephrotoxic nephritis was also decreased when assessed morphologically and by severity of albuminuria. The results demonstrate the power of Ad-IκB-transduced BMDMs to inhibit injury when activated by acute immune-mediated inflammation within the glomerulus. Infiltrating macrophages (mϕ) can cause injury or facilitate repair, depending on how they are activated by the microenvironment. Studies in vitro have defined the roles of individual cytokines and signaling pathways in activation, but little is known about how macrophages integrate the multiple signals they receive in vivo. We inhibited nuclear factor-κB in bone marrow-derived macrophages (BMDMs) by using a recombinant adenovirus expressing dominant-negative IκB (Ad-IκB). This re-orientated macrophage activation so they became profoundly anti-inflammatory in settings where they would normally be classically activated. In vitro, the lipopolysaccharide-induced nitric oxide, interleukin-12, and tumor necrosis factor-α synthesis was abrogated while interleukin-10 synthesis increased. In vivo, fluorescently labeled BMDMs transduced with Ad-IκB and injected into the renal artery significantly reduced inducible nitric oxide synthase and MHC class II expression when activated naturally in glomeruli of rats with nephrotoxic nephritis. Furthermore, although they only comprised 15% of glomerular macrophages, their presence significantly reduced glomerular infiltration and activation of host macrophages. Injury in nephrotoxic nephritis was also decreased when assessed morphologically and by severity of albuminuria. The results demonstrate the power of Ad-IκB-transduced BMDMs to inhibit injury when activated by acute immune-mediated inflammation within the glomerulus. Macrophage infiltration is one of the hallmarks of severe or progressive renal disease, and studies over the past decade provide compelling evidence that they cause tissue injury. The severity of injury correlates with the intensity of the macrophage infiltrate in patients with glomerulonephritis and in experimental models.1Nikolic-Paterson DJ Lan HY Atkins RC Macrophages in immune renal injury.in: Neilson EG Couser WG Immunologic Renal Diseases. Lippincott-Raven, Philadelphia1997: pp 575-592Google Scholar, 2Kluth DC Erwig LP Rees AJ Multiple facets of macrophages in renal injury.Kidney Int. 2004; 66: 542-557Crossref PubMed Scopus (117) Google Scholar Immunohistological studies show that macrophages infiltrating the kidneys of patients and rodents with focal necrotizing glomerulonephritis express MHC class II molecules and inducible nitric oxide synthase, as well as other activation markers typical of a pro-inflammatory phenotype. Purified glomerular macrophages from rats with acute nephritis have the characteristics of classically activated (cytotoxic) macrophages.3Erwig LP Stewart K Rees AJ Macrophages from inflamed but not normal glomeruli are unresponsive to anti-inflammatory cytokines.Am J Pathol. 2000; 156: 295-301Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar Proof of the pathogenetic role of macrophages is provided by macrophage depletion and repletion experiments in rodent models of acute nephritis, which show that depletion abrogates glomerular injury and that this is reversed by adoptive transfer of macrophages. Furthermore, activation of adoptively transferred cells with interferon-γ (IFN-γ) increases their toxicity4Ikezumi Y Atkins RC Nikolic-Paterson DJ Interferon-gamma augments acute macrophage-mediated renal injury via a glucocorticoid-sensitive mechanism.J Am Soc Nephrol. 2003; 14: 888-898Crossref PubMed Scopus (65) Google Scholar while inhibiting JNK, a key pro-inflammatory signaling pathway greatly decreases it.5Ikezumi Y Hurst LA Atkins RC Nikolic-Paterson DJ Macrophage-mediated renal injury is dependent on signaling via the JNK pathway.J Am Soc Nephrol. 2004; 15: 1775-1784Crossref PubMed Scopus (50) Google Scholar However, the intensity of glomerular macrophage infiltration does not always correlate with the severity of injury,6Tam FW Smith J Cashman SJ Wang Y Thompson EM Rees AJ Glomerular expression of interleukin-1 receptor antagonist and interleukin-1 beta genes in antibody-mediated glomerulonephritis.Am J Pathol. 1994; 145: 126-136PubMed Google Scholar, 7Chadban SJ Tesch GH Lan HY Atkins RC Nikolic-Paterson DJ Effect of interleukin-10 treatment on crescentic glomerulonephritis in rats.Kidney Int. 1997; 51: 1809-1817Crossref PubMed Scopus (32) Google Scholar and there is indirect evidence that they contribute to resolution of glomerular inflammation. The adoptive transfer into the kidney of small numbers of macrophages expressing anti-inflammatory cytokines such as interleukin (IL)-4 and IL-10 provides evidence of the effectiveness of anti-inflammatory macrophages in reducing renal inflammation.8Kluth DC Ainslie CV Pearce WP Clarke D Anegon I Rees AJ Macrophages transfected with adenovirus to express IL-4 reduce inflammation in experimental glomerulonephritis.J Immunol. 2001; 166: 4728-4736PubMed Google Scholar, 9Wilson HM Stewart K Brown PAJ Anegon I Chettibi S Rees AJ Kluth DC Bone marrow derived macrophages (BMDM) genetically modified to produce IL-10 reduce injury in experimental glomerulonephritis.Mol Ther. 2002; 6: 710-717Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar This is consistent with studies of inflammation in lung and skin that clearly demonstrate the importance of macrophages for resolution of injury and for tissue repair.10Teder P Vandivier RW Jiang D Liang J Cohn L Pure E Henson PM Noble PW Resolution of lung inflammation by CD44.Science. 2002; 296: 155-158Crossref PubMed Scopus (586) Google Scholar, 11Nagaoka T Kaburagi Y Hamaguchi Y Hasegawa M Takehara K Steeber DA Tedder TF Sato S Delayed wound healing in the absence of intercellular adhesion molecule-1 or L-selectin expression.Am J Pathol. 2000; 157: 237-247Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar These results highlight the heterogeneity of inflammatory macrophages, and insights into how this occurs have been gained from in vitro studies analyzing the different activating factors and downstream intracellular signaling pathways. IFN-γ together with pro-inflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α) induce classical macrophage activation characterized by anti-microbial and cytotoxic properties as part of the host responses to infection or autoimmune disease,12Flavell RA The relationship of inflammation and initiation of autoimmune disease: role of TNF super family members.Curr Top Microbiol Immunol. 2002; 266: 1-9PubMed Google Scholar whereas exposure to IL-4 or IL-13 results in alternatively activated macrophages that attenuate inflammation and promote tissue repair.13Gordon S Alternative activation of macrophages.Nat Rev Immunol. 2003; 3: 23-35Crossref PubMed Scopus (4896) Google Scholar Other stimuli result in different, less well-characterized activation states (reviewed in 14Mosser DM The many faces of macrophage activation.J Leuk Biol. 2003; 73: 209-212Crossref PubMed Scopus (1472) Google Scholar). By contrast, incubation with exogenous IL-10 de-activates macrophages,15Moore KW de Waal Malefyt R Coffman RL O'Garra A Interleukin-10 and the interleukin-10 receptor.Annu Rev Immunol. 2001; 19: 683-765Crossref PubMed Scopus (5487) Google Scholar and IL-10 is an important negative feedback regulator in pro-inflammatory macrophages. Accordingly, macrophages activated by ligation of IL-1 and TNF-α superfamily receptors (including Toll-like receptors), oxidative stress, and UV radiation16Li Q Verma IM NF-kappaB regulation in the immune system.Nat Rev Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3414) Google Scholar rapidly release large amounts of pro-inflammatory mediators, but later, they synthesize large quantities of IL-10.15Moore KW de Waal Malefyt R Coffman RL O'Garra A Interleukin-10 and the interleukin-10 receptor.Annu Rev Immunol. 2001; 19: 683-765Crossref PubMed Scopus (5487) Google Scholar The nuclear factor-κB (NF-κB) signaling pathway plays a central role in the pro-inflammatory macrophage responses to LPS, but it is not known how the subsequent IL-10 response is controlled and, specifically, whether NF-κB is involved. In resting cells, NF-κB is sequestered in the cytoplasm by inhibitor of NF-κB (IκB). Stimulation of signaling pathways leads to activation of IκB kinase complex, which phosphorylates IκB serines at positions 32 and 36. IκB then can be ubiquitinated and degraded by the 26S proteosome, releasing NF-κB, which translocates to the nucleus where it initiates transcription of NF-κB-sensitive genes. The question remains how macrophages with an inhibited NF-κB pathway will respond to the inflammatory environment in vivo and what effect they in turn will have on the progression of injury. Our strategy was to characterize the properties of rat bone marrow-derived macrophages (BMDMs) transduced with a recombinant adenovirus expressing dominant-negative IκB to block NF-κB signaling. The dominant-negative form of IκB has serine 32 and 36 mutated to alanine, so it cannot be phosphorylated and degraded.17Foxwell B Browne K Bondeson J Clarke C de Martin R Brennan F Feldmann M Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumor necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent.Proc Natl Acad Sci USA. 1998; 95: 8211-8215Crossref PubMed Scopus (240) Google Scholar This provides a highly effective biological approach to block NF-κB signaling. We hypothesized that inhibition of NF-κB by transduction with a constitutively active IκB would inhibit the pro-inflammatory consequences of classically activated macrophages while leaving the delayed IL-10 response intact. If this were the case, then NF-κB-inhibited macrophages entering an acutely inflamed glomerulus or other pro-inflammatory environment would respond by expressing IL-10 without prior release of pro-inflammatory mediators and thus might be profoundly anti-inflammatory. Accordingly, we designed the present experiments, first, to ascertain whether inhibition of NF-κB did abrogate the IL-10 response in vitro, and we then used a novel strategy to determine the effects of infusing small numbers of NF-κB blocked cells on injury in rats with nephrotoxic nephritis. The results demonstrate that NF-κB-blocked macrophages release IL-10 after activation, and that such macrophages do not become classically activated after infusion into the kidneys of rats with nephrotoxic nephritis but instead have a dominant anti-inflammatory effect and attenuate injury. Rat BMDMs were isolated by aspiration of the femur and tibia and suspended in culture medium comprising Dulbecco's modified Eagle's medium supplemented with 10% heat inactivated fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin with the addition of 20% L929 conditioned medium produced using a standard protocol;18Lake FR, Noble PW, Henson PM, Riches DW: Functional switching of macrophage responses to tumor necrosis factor-alpha (TNF alpha) by interferons: implications for the pleiotropic activities of TNF alpha. J Clin Invest 199, 93:1661–1669Google Scholar this yields a population of >95% macrophages. For all studies, cells were cultured for 5 days after harvesting, after which time they were transduced with adenovirus. Nephrotoxic serum was produced according to our standard protocol.19Bhan AK Schneeberger EE Collins AB McCluskey RT Evidence for a pathogenic role of a cell-mediated immune mechanism in experimental glomerulonephritis.J Exp Med. 1978; 148: 246-260Crossref PubMed Scopus (139) Google Scholar The recombinant adenoviral vectors adenovirus expressing dominant-negative IκBα (Ad-IκBα) and p65RHD were a gift from Christian Jobin (University of North Carolina) and Joseph Anrather (Harvard Medical School, Boston, MA), respectively, and were prepared as described previously.20Jobin C Morteau O Balfour-Sartor R Specific NF-kappaB blockade selectively inhibits tumour necrosis factor-alpha-induced COX-2 but not constitutive COX-1 gene expression in HT-29 cells.Immunology. 1998; 95: 537-543Crossref PubMed Scopus (125) Google Scholar Briefly, cDNA coding for dominant-negative IκBα and p65RHD were cloned into the pAC.CMV-pLpASR+vector. The resulting pAC.CMV/IκB plasmid plus the purified fragment of ClaI-digested DNA from E1-deleted adenovirus type 5 and p65RHD/pAC.CMV-pLpASR+ vector with the pJM17 recombination plasmid containing the full-length adenoviral genome with a deletion in the E1 region were coinfected into 293 cells (human embryonic kidney cells). Adenovirus clones obtained by limiting dilution in 293 cells were tested for both Ad-IκBα and p65RHD overexpression using Western blotting. Null adenovirus containing no insert (Ad-null), a gift from Dr. F. Graham (Ontario, Canada), was used as control virus throughout the study. All adenoviruses were produced in 293 cells and subsequently purified on a caesium chloride gradient, dialyzed against 10 mmol/L Tris buffer, pH 7.4, with 10% (v/v) glycerol, and stored at −70°C; viral preparations had low or zero levels of endotoxin. Viral titer was determined by plaque assay on human embryonic kidney 293 cells. Transduction was performed in standard medium with 2% fetal calf serum.21Kluth DC Erwig LP Rees AJ Gene transfer into inflamed glomeruli using macrophages transfected with adenovirus.Gene Ther. 2000; 7: 263-270Crossref PubMed Scopus (32) Google Scholar Rat BMDMs uninfected or infected for 48 hours with 100 pfu/cell of Ad-null, Ad-IκB, or p65RHD were stimulated for 2 hours with 100 ng/ml LPS (Sigma-Aldrich, Dorset, UK) and/or 10 ng/ml IFN-γ (BD Biosciences Pharmingen, Cowley, Oxford, UK) and incubated for 15 minutes with ice-cold buffer containing 10 mmol/L Hepes, 10 mmol/L KCl, 0.1 mmol/L EGTA, 0.1 mmol/L EDTA, and 1 mmol/L dithiothreitol supplemented with a cocktail of protease inhibitors with broad specificity for the inhibition of serine, cysteine, aspartic proteases, and aminopeptidases (Sigma-Aldrich). Cells were lysed by adding 10% Nonidet P-40, supernatant was removed, and the pellet resuspended in ice-cold buffer containing 20 mmol/L Hepes, 0.4 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 5% glycerol supplemented with protease inhibitors. The mixture was incubated for 60 minutes, and supernatants (nuclear extract) were removed by centrifugation. Equal amounts of nuclear extracts were incubated for 10 minutes at 37°C with [γ-32P]ATP-radiolabeled NF-κB oligonucleotide (5′-AGT TGA GGG GAC TTT CCC AGG C-3′ 3′-TCA ACT CCC CTG AAA GGG TCC G-5′) (Promega, Southampton, United Kingdom), and the DNA protein complexes were separated on a 5% polyacrylamide gel. Normal BMDMs and BMDMs transduced with Ad-null or Ad-IκB for 48 hours were stimulated for 24 hours with 100 ng/ml LPS (Sigma-Aldrich) and/or 10 ng/ml IFN-γ (BD Biosciences Pharmingen) in 1-ml wells. Supernatants were removed, and cells were harvested and counted. The concentration of cytokines in the supernatant was determined by capture enzyme-linked immunosorbent assay using kits specific for TNF-α, IL-10, active TGF-β, (BD Pharmingen) and IL-12 (Biosource International California). Generation of NO by macrophages was assessed by nitrite production assayed using a Greiss reaction22Erwig LP Kluth DC Walsh GM Rees AJ Initial cytokine exposure determines macrophage function and renders them unresponsive to other cytokines.J Immunol. 1998; 161: 1983-1988PubMed Google Scholar without reduction of nitrite. To detect cell surface expression of MHC II or CD86, BMDMs were treated with IFN-γ and scraped off wells, and 4 × 105 cells/ml were incubated for 1 hour with 2 μg/ml fluorescein isothiocyanate (FITC)-conjugated monoclonal to either mouse anti-rat MHC class II Ia IgG (Serotec, MCA46FT) or mouse anti rat CD86 (B7-2, 555018; BD Pharmingen) and analyzed by flow cytometry Specific labeling was determined by comparing with nonspecific staining using a FITC-labeled isotype-matched control mAb. Expression of MHC class II or CD86 was determined in terms of specific mean fluorescence relative to isotype control antibody. In each experiment, 10,000 events per sample were collected. Inbred male Sprague-Dawley rats (200–250 g) (Harlan, Bicester, Oxon) were pre-immunized with subcutaneous injection of 1 mg of rabbit IgG (Sigma-Aldrich) in Freund's complete adjuvant and injected intravenously 1 week later with rabbit nephrotoxic serum. This results in macrophage infiltration, acute glomerular injury, and crescent formation. Normal and transduced macrophages were labeled with a red fluorescent membrane label, PKH-26GL21Kluth DC Erwig LP Rees AJ Gene transfer into inflamed glomeruli using macrophages transfected with adenovirus.Gene Ther. 2000; 7: 263-270Crossref PubMed Scopus (32) Google Scholar and harvested into serum-free media immediately before injection. Labeled macrophages were injected directly into the renal artery 24 hours after induction of NTN by performing a midline laparotomy and isolating the left renal artery. The cells were injected into the artery over 1 to 2 minutes, and renal blood flow re-established in less than 5 minutes. Groups of at least six rats were killed 2, 3, or 6 days after disease induction. The proportion of glomerular macrophages that had been adoptively transferred was measured in two ways. First the proportion of NFκB-blocked macrophages was calculated using immunohistology by relating the number of fluorescent PKH 26GL-labeled (adoptively transferred) macrophages to the total number of ED1-positive glomerular macrophages in 50 consecutive glomeruli. The proportion was also calculated using flow cytometry by determining the proportion of CD11b-positive cells (identified using OX42, Serotec MCA275FT) that also were labeled with PKH 26L. Rats were housed in metabolic cages for 14 hours each day for up to 6 days after disease induction for urine collection. Urine albumin concentration was determined using rocket electrophoresis21Kluth DC Erwig LP Rees AJ Gene transfer into inflamed glomeruli using macrophages transfected with adenovirus.Gene Ther. 2000; 7: 263-270Crossref PubMed Scopus (32) Google Scholar and albuminuria calculated as the total albumin excreted over 24 hours. Sections of renal tissue fixed in methyl Carnoy's fixative were paraffin embedded, and 3-μm sections were cut before routine staining with hemotoxylin and eosin (H&E), methenamine silver, and Acid Fuchsin Orange G (to highlight necrotic lesions and fibrin deposition) and immunohistochemistry. Tissue sections were examined histologically for severity of glomerular injury by a pathologist blinded to the origin of the sample, using a standard scoring system as follows; necrosis (0, absent; 1, focally present over single glomerular segments; 2, widespread) and sclerosis (0, absent; 1, mild; 2, moderate; 3, severe). Glomerular numbers affected by crescents were counted per 40 glomeruli. Total cellularity was quantified (nuclear counts/glomerular cross-section, mean of 20 glomeruli) regardless of the nature of the cells as infiltrating macrophage numbers were assessed independently. Glomerular macrophage infiltration was assessed in tissue sections that were deparaffinized in xylene and rehydrated in graded alcohol before being incubated with an antibody to CD68 (ED1 Serotec, MCA341R) or MHC class II (OX6, Serotec, MCA46R). The primary antibodies were visualized by alkaline phosphatase-labeled rabbit anti-mouse and mouse APAAP (Dako) and developed using Fast red as a substrate. The number of ED1 and MHC class II-positive cells were counted in 50 glomeruli at ×200 magnification by two independent observers, and the average number per glomerulus was calculated. Mesangium stained weakly for MHC class II in some cases, but strong positive staining was only observed in macrophages as identified by a double stain with ED1. Kidney was cut into pieces of <1 mm3 in ice-cold Paris buffer (20 mmol/L Tris/HCl, 125 mmol/L NaCl, 10 mmol/L KCl, 10 mmol/L sodium acetate, and 5 mmol/L glucose) and tissue sieved through 250- and 150-μm sieves, and glomeruli were collected on a 63-μm filter. For visualization of the fluorescent macrophages within glomeruli, the isolated glomeruli were labeled with anti-rabbit IgG FITC (Sigma-Aldrich) that binds to the rabbit IgG from the nephrotoxic serum deposited on the glomerular basement membrane, and the number of PKH-26GL-positive, fluorescent cells in 100 glomeruli was counted. Isolated glomeruli were washed in Hanks' balanced salt solution (HBSS) with calcium and magnesium (Sigma-Aldrich) and then incubated for 10 minutes at 37°C in 4 ml of prewarmed HBSS containing 0.5 mg/ml trypsin, 1 mg/ml collagenase (Type I), and 0.1 mg/ml DNase (Sigma-Aldrich). After washing in HBSS without calcium and magnesium, the partially digested glomeruli were incubated in 2 mmol/L EDTA, disodium salt in the same buffer for 10 minutes at 37°C. For final dissociation, the cell preparation was passed five times through a 23-gauge needle, and cells were washed in HBSS and counted. MHC class II-positive macrophages were detected by incubating 8 × 105 isolated glomerular cells/ml for 1 hour with 4 μg/ml of FITC-conjugated monoclonal to anti-rat/mouse I-A IgG (Serotec, MCA46FT). For detection of inducible nitric oxide synthase (iNOS), cells were fixed and permeabilized by Cytofix/cytoperm reagent (BD Pharmingen), blocked with 10% goat serum (Sigma-Aldrich), and incubated for 1 hour with 2 μg/ml anti rat iNOS23Helfrich MH Evans DE Grabowski PS Pollock JS Ohshima H Ralston SH Expression of nitric oxide synthase isoforms in bone and bone cell cultures.J Bone Miner Res. 1997; 12: 1108Crossref PubMed Scopus (152) Google Scholar followed by FITC-labeled goat anti-rabbit IgG (Sigma-Aldrich) for 1 hour. Specific labeling was compared with nonspecific staining using a FITC-labeled isotype-matched control antibody. Single-cell preparations of glomeruli were analyzed on a FACSCalibur and using CellQuest software (BD Biosciences). A gate was set to include all PKH-26L-labeled cells, and the FL1 fluorescence from these (ie, those MHC class II or iNOS positive) was displayed as a histogram (Figure 4A). Results were expressed as the percentage of PKH-26L-labeled cells that were MHC class II or iNOS positive relative to isotype control antibody. Results are presented as mean ± SD, and differences between groups of cells or animals were tested using a two-way analysis of variance followed by a multiple range test (Tukey analysis) or a Mann-Whitney nonparametric ranking test. Our previous work has shown rat BMDMs are transduced with 50 to 150 pfu/cell recombinant adenoviruses with high efficiency and little toxicity.8Kluth DC Ainslie CV Pearce WP Clarke D Anegon I Rees AJ Macrophages transfected with adenovirus to express IL-4 reduce inflammation in experimental glomerulonephritis.J Immunol. 2001; 166: 4728-4736PubMed Google Scholar, 9Wilson HM Stewart K Brown PAJ Anegon I Chettibi S Rees AJ Kluth DC Bone marrow derived macrophages (BMDM) genetically modified to produce IL-10 reduce injury in experimental glomerulonephritis.Mol Ther. 2002; 6: 710-717Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 21Kluth DC Erwig LP Rees AJ Gene transfer into inflamed glomeruli using macrophages transfected with adenovirus.Gene Ther. 2000; 7: 263-270Crossref PubMed Scopus (32) Google Scholar We confirmed this was true for Ad-IκB transduction because of reports that NF-κB inhibition can increase sensitivity to apoptosis.24Wang CY Mayo MW Baldwin AS TNF-and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB.Science. 1996; 274: 784-787Crossref PubMed Scopus (2522) Google Scholar Viability was not significantly altered when BMDMs were transduced with Ad-IκB at these concentrations (Table 1). Expression of the dominant-negative IκB protein, assessed by Western blotting, reached a maximum by 48 hours, and the expression was not influenced by LPS or IFN-γ. Consequently, a viral dose of 100 pfu/cell was used in subsequent transductions.Table 1Properties of Ad-IκB-Transduced MacrophagesPfu/cellPercentage of cells transducedMean fluorescent intensityViability5064 ± 412299 ± 810089 ± 6143100 ± 615086 ± 813798 ± 1120072 ± 514989 ± 7Transduction efficiency, mean fluorescence intensity, and viability of Ad-IκB-transduced bone marrow-derived macrophages as determined by flow cytometry using an anti-rat IκB antibody and by propidium iodide, respectively. Maximum transduction efficiency with minimum cell death was achieved with 100 pfu/cell. Open table in a new tab Transduction efficiency, mean fluorescence intensity, and viability of Ad-IκB-transduced bone marrow-derived macrophages as determined by flow cytometry using an anti-rat IκB antibody and by propidium iodide, respectively. Maximum transduction efficiency with minimum cell death was achieved with 100 pfu/cell. The ability of dominant-negative IκB to suppress NF-κB nuclear translocation was confirmed on electrophoretic mobility shift assay (EMSA). We investigated the presence of Rel/NF-κB in the nuclear extract of either unstimulated or LPS and IFN-γ/LPS-stimulated cells. LPS or IFN-γ/LPS stimulation resulted in an increased Rel/NF-κB translocation to the nucleus (Figure 1). Confirmation that the identified EMSA band was p65 was obtained by transducing macrophages with an adenovirus expressing a dominant-negative form of p65. This gives an intense p65 band that was supershifted by pre-incubation with anti-p65 antibodies. In contrast, transduction of macrophages with Ad-IκB resulted in a marked decrease in nuclear NF-κB that was not increased by either LPS or a combination of IFN-γ/LPS stimulation thus indicating sequestration of RelA/NF-κB in the cytoplasm. As expected, NO, TNF-α, and IL-12 responses were profoundly inhibited in Ad-IκB-transduced macrophages (Table 2), demonstrating the effectiveness of blocking pro-inflammatory effects. Also, transduction with either Ad-IκB or Ad-null did not inhibit LPS-induced IL-10 synthesis (Table 2). In fact the response was amplified in Ad-IκB-transduced cells. TGF-β synthesis did not differ in control and Ad-IκB-transduced BMDMs.Table 2Effect of Ad-IκB Transduction on Nonstimulated and LPS-Induced TNF-α, IL-12, Nitrite, IL-10, and Active TGF-β Expression by Rat Bone Marrow-Derived MacrophagesLPSNontransducedAd-nullAd-IκBNontransducedAd-nullAd-IκBTNF-α (pg/ml)28 ± 2250 ± 31*P < 0.05 compared with nontransduced cells.128 ± 11*P < 0.05 compared with nontransduced cells.1342 ± 1211721 ± 165572 ± 58†P < 0.05 compared with nontransduced and Ad-null transduced cells.IL-12 (pg/ml)BLDBLDBLD172 ± 18198 ± 21100 ± 8†P < 0.05 compared with nontransduced and Ad-null transduced cells.Nitrite (μmol/L)12 ± 239 ± 2*P < 0.05 compared with nontransduced cells.27 ± 8*P < 0.05 compared with nontransduced cells.166 ± 11176 ± 1362 ± 7†P < 0.05 compared with nontransduced and Ad-null transduced cells.IL-10 (pg/ml)88 ± 1064 ± 4130 ± 19*P < 0.05 compared with nontransduced cells.1607 ± 1561718 ± 1322058 ± 189†P < 0.05 compared with nontransduced and Ad-null transduced cells.TGF-β (pg/ml)3685 ± 3333583 ± 3433201 ± 2273473 ± 5102349 ± 2502577 ± 390Ad-IκB transduction resulted in the inhibition of NO release and TNF-α and IL-12 production by macrophages in response to LPS stimulation while IL-10 concentration was elevated. BLD, below limit of detection. Results are means ± SD (n = 5) and were compared by ANOVA followed by a multiple range test (Tukey analysis).* P < 0.05 compared with nontransduced cells.† P < 0.05 compared with nontransduced and Ad-null transduced cells. Open table in a new tab Ad-IκB transduction resulted in the inhibition of NO release and TNF-α and IL-12 production by macrophages in response to LPS stimulation while IL-10 concentration was elevated. BLD, below limit of detection. Results are means ± SD (n = 5) and were compared by ANOVA followed by a multiple range test (Tukey analysis). Next, we assessed the ability of Ad-IκB transduction to block classical macrophage activation by the combination of IFN-γ and LPS. The increase in IL-12 was significantly attenuated in Ad-IκB-transduced cells (Figure 2A). Interestingly, the IL-12 response substantially increased by 48 hours, even though levels remained significantly less than those of normal BMDMs and Ad-null-transduced cells. Ad-IκB also blocked the normal IFN-γ-induced increase in NO generation (Figure 2B), MHC class II expression (Figure 2C), and CD-86 expression (Figure 2
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