Abstract
Granulocyte macrophage–colony-stimulating factor (GM-CSF), released from alveolar macrophages (AM), is an important regulator of eosinophil, T cell, and macrophage function and survival. We determined the mechanisms of GM-CSF regulation in AM from normal volunteers activated by lipopolysaccharide (LPS) by examining the role of nuclear factor-κB (NF-κB), and of p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK-1). PD 098059 (10 μM), an inhibitor of upstream activator of MKK-1, inhibited GM-CSF expression, but the expression of GM-CSF was not inhibited by SB 203580 (10 μM), an inhibitor of p38-MAP kinase. Phosphorylation of extracellular signal–regulated kinase-1 (ERK-1), ERK-2, and p38 MAP kinase by LPS were demonstrated on Western blot analysis. LPS increased NF-κB:DNA binding as examined by electrophoretic mobility shift assay, but this was not suppressed by PD 098059 or by SB 203580. LPS induced an increase in NF-κB activation as examined by p50 translocation assay without suppression by PD 098059 or by SB 203580. SN50 (100 μM), an inhibitor of NF-κB translocation and the specific IKK-2-Inhibitor (AS602868; 10 μM), also prevented GM-CSF expression and release induced by LPS, indicating that GM-CSF release is NF-κB–dependent. PD 098059, but not SB 203580, inhibited LPS-induced histone acetyltransferase (HAT) activity, indicating chromatin modification. Furthermore, AS602868 and SN 50 suppressed LPS-induced HAT activity. TSA (10 ng/ml), an inhibitor of histone deacetylase (HDAC), reversed the inhibitory effect of PD 098059, SB 203580, SN 50 and AS602868 on GM-CSF release. GM-CSF expression and release in AM is controlled by NF-κB activation, and this is modulated by phosphorylation of MKK-1 and p38 MAP kinase acting on histone acetylation.
Human alveolar macrophages (AM) originate from multipotent progenitor cells in bone marrow and serve physiologic roles in host immunity, wound healing, and inflammation (1). Upon activation, AM release several inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), leukotriene B4, and granulocyte macrophage–colony-stimulating factor (GM-CSF) that attract and activate neutrophils, eosinophils, and blood monocytes. GM-CSF is of particular interest because it regulates inflammatory cell function and survival (2, 3), in addition to its inhibition of apoptosis of human neutrophils (4, 5), involvement in the accumulation of neutrophils in the airways and enhancement of eosinophil survival (4, 6). GM-CSF augments leukotriene C4 and superoxide anion generation from eosinophils (7), and promotes the synthesis of TNF-α, thus playing an important role in the amplification of inflammatory processes. Furthermore, GM-CSF induces the proliferation and differentiation of monocytes into distinct subsets of macrophages (8) and act as a pivotal factor for the development of alveolar macrophages in lung (9).
The intracellular signaling pathways involved in the regulation of GM-CSF expression in alveolar macrophages are unclear. In epithelial cells, GM-CSF generation is primarily regulated at the level of gene transcription by transcription factors activated via phosphorylation (10). One ubiquitous transcription factor of particular importance in immune and inflammatory responses is nuclear factor κB (NF-κB), which increases the rate of transcription of the inflammatory genes and therefore increase formation of mRNA and protein (11). GM-CSF generation induced by lipopolysaccharide (LPS) in human blood monocytes has been shown to be NF-κB–independent (12). There is evidence that transcriptional activation of monocytes and macrophages changes during recruitment from peripheral blood to the airways.
The MAP kinase cascade is involved in signaling from the cytosol to the nucleus and is activated by diverse stimuli that include growth factors, cytokines, and endotoxins, as well as by many pharmacologic signaling agonists (13). In cells such as blood monocytes, LPS induces the phosphorylation of extracellular signal–regulated kinase (ERK)-1, ERK-2, and p38 mitogen-activated protein (MAP) kinase. The MAP kinase cascade may cause activation of a small subset of genes, first by causing phosphorylation of specific transcription factors such as CREB binding protein (CBP), and second, by modifying the phosphorylation and/or acetylation status of histones, thereby modulating the chromatin milieu of these genes (14) (15). Acetylation of histone residues results in unwinding of DNA coiled around the histone core, thus opening up the chromatin structure, which allows transcription factors and RNA polymerase II to bind more readily, thereby increasing transcription. Repression of gene expression by dephosphorylation of MAP kinases may occur through inhibition of histone acetylation or by increasing histone deacetylation. In human epithelial cells, under conditions of maximal histone H4 acetylation, NF-κB–mediated GM-CSF activation is increased (16).
The precise mechanism involved in switching chromatin from active to inactive conformations are not fully understood, but one possible mechanism involves the post-transcriptional modification of chromatin-associated proteins. In addition to activating transcription factors directly by phosphorylation, MAP kinases may also play a role in modifying the chromatin environment of the targeted genes. The purpose of this study was to determine the role of the MAP kinase kinase (MKK)-1 and p38 MAP kinase signaling cascades in LPS-induced GM-CSF generation from human alveolar macrophages, and to examine the involvement of NF-κB and of histone acetylation. We therefore studied the effects of selective inhibitors of the downstream MKK-1 inhibitor of ERK-1 and -2, PD 098059, and of p38 MAP kinase, SB 203580. We examined the role of NF-κB by using the synthetic cell-permeable peptide containing a domain of the peptide from fibroblast growth factor linked to the nuclear localization sequence of the p50 subunit of NF-κB, SN50 (17), and additionally the specific IKK-2 inhibitor, AS602868 (18).
Sixteen normal nonatopic nonsmoking volunteers (11 male, 5 female; mean age: 27 yr), who had no history of asthma or of any respiratory disease with normal lung function and airway responsiveness to methacholine (PC20 > 16 mg/ml) were included in this study. All volunteers underwent fiberoptic bronchoscopy and bronchoalveolar lavage after giving written informed consent. The study was approved by the Ethics Committee of the Royal Brompton Hospital, London, and of the University of Cologne, Germany.
After an overnight fast, subjects were sedated with intravenous midazolam (5–10 mg). Oxygen (3 liters/min) was administered via nasal prongs, and oxyen saturation was monitored by digital pulse oximetry. Using local anesthesia with lidocaine (2% wt/vol) to the upper airways and larynx, a fiberoptic bronchoscope (Olympus BF10, Hamburg, Germany) was passed through the nasal passages into the trachea. The bronchoscope was wedged in the right middle lobe and 4 × 60 ml aliquots of prewarmed sterile 0.9% NaCl solution were instilled. This solution was aspirated through the bronchoscope, collected in prechilled glass bottles, and stored on ice. Bronchoalveolar lavage (BAL) recovery was between 120 and 200 ml.
The BAL fluid (BALF) was filtered through sterile gauze to exclude mucus plugs and was then centrifuged at 1,000 × g for 10 min at 4°C to obain a cell pellet. The cell pellet was washed once in 50 ml of Ca2+/Mg2+ free Hanks' balanced salt solution. The cells were counted on a hemocytometer (Neubauer chamber; Brand, Wertheim, Germany) slide using a Kimura counterstain and viability assessed by trypan blue exclusion. Cytospins were performed using 2.5 × 104 cells/slide, and stained with May-Grünwald-Giemsa to obtain differential cell counts. The remaining cells were resuspended in RPMI 1640 medium supplemented with 10% (vol/vol) fetal calf serum (FCS), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μ/ml streptomycin. A quantity of 3 × 106 macrophages/well were plated onto 6-well plates, and 1 × 106 macrophages/well were plated onto 12-well plates and allowed to adhere for a minimum of 3 h in a humidified incubator in 95% air, 5% CO2 (vol/vol), at 37°C. Nonadherent cells were removed by washing with RPMI 1640 medium, leaving the adherent macrophages, which were > 99% pure, as assessed by staining and morphologic analysis. The macrophages were either harvested with a cell scraper or were stimulated, cultured, and then recovered.
The total cell counts recovered from these volunteers were 20.5 ± 3.6 × 106 cells, with a differential count of 90.9 ± 2.3% macrophages, 3.2 ± 0.8% neutrophils, 0.9 ± 0.1% eosinophils, and 3.9 ± 1.7% lymphocytes. Cells were > 85% viable, as assessed by trypan blue exclusion.
Alveolar macrophages were cultured at 37°C in a humified atmosphere with 5% CO2 in RPMI 1640 medium (Sigma, Munich, Germany) supplemented with 10% (vol/vol) FCS (Sigma), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μ/ml streptomycin. After 12 h, cells were stimulated in fresh RPMI medium as above at 1 × 106 cells/ml for enzyme-linked immunosorbent assay (ELISA) for 24 h and with RPMI medium in the absence of FCS at 3 × 106 cells/ml for electrophoretic mobility shift assay (EMSA) and Western blotting for 1 h. The ERK1/2 inhibitor, PD 098059 (10 μM), and the p38MAPK-inhibitor SB 203580 (10 μM; Calbiochem-NovaBiochem UK Ltd, Nottingham, UK), the NF-κB-inhibitor SN 50 (100 μM; BioMol, Hamburg, Germany) and the IKK-2-Inhibitor (10−5 M and 10−6 M) AS602868 (Serono, Basel, Switzerland) were added 30 min before stimulation with LPS in different concentrations and in a time-dependent manner (Sigma). Drugs were dissolved in dimethyl sulfoxide (Me2SO) or distilled water and were diluted to final concentrations of < 0.1% (vol/vol). Me2SO alone had no effect on activation of NF-κB or cytokine transcription or expression (data not shown).
Extracts were prepared from alveolar macrophages according to the method of Osborn and coworkers (19). Cells were washed twice with ice-cold Hanks' balanced salt solution before resuspension in 10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1% vol/vol Nonidet P40 (NP-40), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM dithiothreitol (DTT) (Buffer A). After a 2-min incubation on ice, nuclei were separated by centrifugation at 1,000 × g for 10 min. Supernatants (cytoplasmic extracts) were retained. For EMSA, nuclei were resuspended in 20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% vol/vol glycerol, 0.5 mM PMSF, and 1 mM DTT (Buffer B) and incubated on ice for 60 min with vigorous mixing. Nuclear debris was removed by centrifugation, and supernatants (nuclear extracts) were diluted 4-fold in 20 mM HEPES, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, and 1 mM DTT (Buffer C).
Nuclear proteins (5 μg) were used in binding reactions as described originally by Osborn and coworkers (19). Double-stranded oligonucleotides encoding the consensus target sequence of NF-κB (5′-AGT TGA GGG GAC TTT CCC AGG-3′) were end-labeled using [γ-32P]-ATP and T4 polynucleotide kinase. Nuclear proteins from each sample were incubated with 5 μl binding buffer (20% [vol/vol] glycerol, 250 mM NaCl, 2.5 mM EDTA, 2.5 mM DTT, 50 mM TRIS-HCl, pH 7.5, 5 mM MgCl2, 0.4 mg/ml sonicated salmon sperm DNA) for 20 min on ice. Then, 0.0175 pmol of radiolabeled oligonucleotide was added and the volume made up to 25 μl with Buffer C. This was incubated for 45 min on ice. Specificity was determined by the prior addition of a 100-fold excess of unlabeled competitor consensus oligonucleotide. To confirm the identity of the components of the retarded complexes, supershift experiments were conducted, using anti-p50 and anti-p65 human antibodies (Promega) at 0.4 μg/ml 30 min before the addition of radiolabeled oligonucleotide. Reactions were separated on 6% nondenaturing acrylamide gels in 0.25× Tris-buffered EGTA. Gels were vacuum-dried, and protein-DNA complexes were visualized by autoradiography at −70°C using Kodak X-OMAT film (Protein Databases Inc., New York, NY). The retarded bands were quantified by laser densitometry. Band density measurements were expressed as NF-κB per microgram of protein loaded.
To detect and quantify NF-κB activation in alveolar macrophages, the translocation of p50, the subunit of NF-κB was assayed using a quantitative sandwich enzyme immunoassay kit (Trans-AM; Active Motif, Rixensart, Belgium) with a patented technology to attach an oligonucleotide containing an NF-κB consensus binding site to a 96-well plate. The activated NF-κB contained in nuclear extracts specifically binds to this oligonucleotide. By using an antibody directed against the NF-κB p50 subunit, the NF-κB complex bound to the oligonucleotide is detected, according to the instruction manual. In short, 5 μg of nuclear sample fractions were diluted in lysis buffer and incubated for 1 h at room temperature. After washing the plates three times, 100 μl of diluted primary antibody (1:1,000 dilution in 1× binding buffer) was added to all wells and incubated for 1 h at room temperature. After three times washing, 100 μl of diluted horseradish peroxidase (HRP)-conjugated antibody (1:1,000 dilution in 1× antibody binding buffer) to all wells being used and incubated for 1 h at room temperature. Then the plate was washed again three times before adding 100 μl of developing solution to all wells and incubated for 2–10 min at room temperature. The blue color development in the sample and positive control wells was monitored until the reaction was stopped by adding 100 μl of stop solution (0.5 M H2SO4). NF-κB p50 translocation was measured colorimetrically at 450 nm and was quantified by interpolation from a standard curve.
For the detection of acetyl residues on an acetylated histone H4 substrate peptide, we used an active histone acetyltransferase (HAT) enzyme according to the instruction manual from Upstate (HAT assay kit, Cat # 21746; Biomol, Hamburg, Germany). After pre-coating each well of the ELISA plate with 100 μl of 1 μg/ml reconstituted histone H4 and a reference standard of acetylated and unmodified peptides, the plate was incubated overnight at 4°C. The wells were washed five times with TBS before adding 200 μl of 3% bovine serum albumin (BSA) and incubated for 30 min at 30°C. After washing the wells five times, 50 μl of HAT reaction cocktail per well was added (10 μl 5× HAT assay buffer, 10 μl 500 μM Acetyl-CoA, 10 μg of cell extracts and add to 50 μl with sterile water) before incubation for 45 min at room temperature. After washing the wells five times, 100 μl of 1:250 anti–acetyl-Lysine ELISA grade in TBS containing 3% BSA was added and incubated for 90 min at room temperature. After washing the wells five times with TBS 1:1,000 diluted anti-rabbit IgG, HRP conjugate in TBS containing 3% BSA was added to each well and incubated for 30 min at room temperature. After washing five times with TBS, 100 μl of the TMB substrate mixture was added to each well and incubated for up to 10 min at room temperature before addition of 50 μl of 1 M sulfuric acid to each well to stop the HRP reaction and reading the plate colorimetrically on a wavelength of 450 nm and 570 nm to substract the 570 nm values from the 450 nm values to remove any well-to-well plate variation.
GM-CSF was assayed using a quantitative sandwich enzyme immunoassay technique. Ninety-six-well plates (NUNC Inc., Weisbaden, Germany) were coated overnight at room temperature with 50 μl of a rat anti-human GM-CSF monoclonal antibody (2 μg/ml) (R&D Systems, Abingdon, UK). After washing with TBS-Tween-20, the antibody was blocked with PBS/10% FCS (300 μl/well) for 2 h. GM-CSF standard (rh GM-CSF; R&D Systems) and samples were added to the plate overnight at room temperature and washed with TBS-Tween-20 four times. A biotinylated secondary anti-human GM-CSF antibody (R&D Systems) (100 μl of 2 μg/ml) in PBS/10% FCS was added for 1 h followed by 1:400 avidin-peroxidase solution (100 μl). After washing, GM-CSF was measured colorimetrically at 405 nm and quantified by interpolation from a standard curve. The lower limit of detection was 16 pg/ml.
The activation status of ERK-1 and ERK-2 was assessed by Western immunoblot analysis using antibodies that recognize the dual phosphorylated (activated) form of the enzymes. After treatment, macrophages were lysed in lysis buffer (20 mM Tris base, pH 7.4; 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, 50 mM NaCl, 5 mM EDTA), supplemented with the proteinase inhibitors PMSF (500 μM), Na-orthovanadate (2 mM), leupeptin (10 μg/ml), aprotinin (25 μg/ml), pepstatin (10 μg/ml), NaF (1.25 mM), and Na-pyrophosphate (1 mM). Insoluble protein was removed by centrifugation at 12,000 × g for 5 min and aliquots of the resulting supernatant were diluted 1:4 in Laemelli buffer (62.5 mM Tris-HCl, 10% vol/vol glycerol, 1% wt/vol SDS, 1% β-mercaptoethanol, 0.01% wt/vol bromophenol blue, pH 6.8) and boiled for 5 min. Denatured proteins (40 μg) were subsequently separated by SDS/PAGE upon 10% vertical slab gels and transferred to Hybond ECL membranes (Amersham, Buckinghamshire, UK) in blotting buffer (Tris base 50 mM, pH 8.3; glycine 192 mM) supplemented with 20% vol/vol methanol. The nitrocellulose was incubated for 1 h in TBS-Tween-20 (25 mM Tris-base, 150 mM NaCl, pH 7.6 and 0.1% Tween 20, 10% [wt/vol] nonfat milk and incubated 2 h in TBS-Tween 20 containing 3% BSA and either primary antibodies raised against pERK1, pERK2, phospho-p38MAPK, or β-actin (diluted 1:1,000). Following 3 × 10-min washes in TBS-Tween 20, the membranes were incubated for 60 min with a goat anti-rabbit peroxidase–conjugated IgG antibody diluted 1:5,000 in TBS-Tween 20 supplemented with 1% nonfat milk for ERK-1 and -2 and p-p38MAPK, and for 60 min with a rabbit, anti-mouse peroxidase–conjugated IgG antibody diluted 1:4,000 in TBS-Tween 20 supplemented with 5% nonfat milk for actin and then washed again (3 × 10 min). Antibody-labeled proteins were subsequently visualized by enhanced chemiluminescence (Amersham). Relevant bands were quantified by laser-scanning densitometry and normalized to the house-keeping protein, β-actin.
HAT assays were performed using a modified method of Ogryzko and colleagues (20). Twenty microliters of free core histone solution extracted from alveolar macrophages (final amount 10 μg) and 30 μl of cell extraction were incubated. Reactions were initiated by the addition of 0.25 μCi of [3H] acetyl-CoA (5 Ci/mmol) (Amersham) and peformed for 45 min at 30°C. After incubation, the reaction mixture was spotted onto Whatman p81 phosphocellulose filter paper (Whatman) and washed for 30 min with 0.2 M sodium carbonate buffer (pH 9.2) at room temperature with 2–3 changes of the buffer, then washed briefly with acetone. The dried filters were counted in a liquid scintillation counter.
RNA was extracted using the Qiagen kit (RNeasy Mini kit; Qiagen, Hilden, Germany), according to the manufacturer's instructions and purity assessed by spectrophotometry. RNA was denatured at 65°C for 10 min. Reverse transcription of 1 μg of total RNA was performed using AMV-reverse transcriptase (15 U), 10 mM of dATP, dCTP, dGTP and dTTP, oligo dT15 primer (0.2 μg), RNase inhibitor (30 U), and 5× AMV buffer in a total volume of 10 μl (Promega, Southampton, UK). The remaining ingredients were then added as a master mix, and samples were incubated at 42°C for 60 min followed by 4 min at 90°C. The cDNA was subsequently diluted to a final volume of 60 μl in nuclease-free water. For PCR, 5 μl of the cDNA solutions were used. PCR was performed using 0.5 μg/μl of forward and reverse primers, dATP, dGTP, dTTP, and dCTP, at a concentration of 10 mM each. Taq polymerase (0.5 U), and buffer A (all Promega) in a final volume of 20 μl set for 30 cycles for GM-CSF and 24 cycles for GAPDH at a denaturing temperature of 94°C for 30 s, specific annealing temperature of 60°C for GAPDH and 55°C for GM-CSF, and extension temperature of 72°C for 30 s. Primers for GAPDH were 5′-TCTAGACGGCAGGCTAGGTCCACC and 3′-CCACCCATGGCAAATTCCATGGCA, giving a product of 598 base pairs. GM-CSF primers used were from R&D Systems, giving a product of 261 base pairs. PCR was performed in a Techne multiwell thermocycler (Techne, Cambridge, UK). The number of cycles was chosen after determination of the linear phase of the product amplification curve from serial sampling with increasing cycles of amplification. Products were distinguished by electrophoresis on a 2% agarose, ethidium bromide-stained gel and then visualized using ultraviolet luminescence.
Data are represented as mean ± SEM. Data were analyzed by Student's t test for paired data or by one-way ANOVA/Bonferroni multiple comparison test. The null hypothesis was rejected when P < 0.05.
The basal generation of GM-CSF in human alveolar macrophages was below the limit of detection of the assay. The GM-CSF product was undetectable until 24 h of stimulation without significant differences in GM-CSF production at 10 ng/ml and 1 μg/ml of LPS. Similarly, GM-CSF mRNA expression reached a maximum after 12 h of LPS stimulation without differences in gene expression between 10 ng/ml and 1 μg/ml of LPS. In subsequent experiments, we therefore used LPS at 10 ng/ml.
PD 098059 (10 μM), an inhibitor of the upstream activator of ERK-1 and ERK-2, suppressed LPS-induced GM-CSF release by 76.7 ± 7.0% (P < 0.01) (Figure 1A)
Figure 1. Inhibitory effect of PD 098059 (10 μM), SB 203580 (10 μM), SN 50 (100 μM), and AS602868 (10−5 M; 10−6 M) on LPS-induced GM-CSF generation and expression. GM-CSF release at 24 h was measured by sandwich ELISAs (A). Data points represent the mean ± SEM of 6–10 determinations using alveolar macrophages from different donors. LPS-induced GM-CSF mRNA expression was measured (B and C). B shows a representative gel and C shows the mean data of 4–6 independent experiments. *P < 0.05 and **P < 0.01 compared with LPS alone.
[More] [Minimize]SN 50 (100 μM) prevented LPS-induced generation of GM-CSF by 83.5 ± 9.5% (Figure 1A). SN 50 decreased GM-CSF mRNA by 69.0 ± 23.6% (Figures 1B and 1C). SN 50 (100 μM) had no cytotoxic effect on cell viability as measured by methylthiazol tetrazolium (MTT) assay. These data are consistent with an inhibition of GM-CSF release by 88.5 ± 4.7% and by 38.9 ± 10.7% in the presence of AS602868 at 10−5 M (P < 0.01) and 10−6 M (NS), respectively (Figure 1A).
The time-dependent phosphorylation of ERK-1, ERK-2, and p38 MAP kinase in LPS-stimulated human alveolar macrophages is shown in Figure 2
Figure 2. Time-course of LPS-induced ERK-1, ERK-2, and p38MAPK phosphorylation was determined by Western blotting. A representative Western blot of ERK-1 and ERK-2 is shown in A, with the mean ± SEM of four experiments of alveolar macrophages from four different donors in B. C and D show a representative Western blot of p38 MAPK and the mean ± SEM of four experiments from alveolar macrophages of four different donors, respectively. E shows a representative Western blot of LPS-induced increase in phosphorylation of ERK-1, ERK-2 and dephosphorylation of ERK-1 and ERK-2 in the presence of PD 098050 (10 μM), whereas F shows a representative Western blot of LPS-induced increase in phosphorylation of p38MAPK and the inhibitory effect of p38MAPK phosphorylation by SB 203580 (10 μM).
[More] [Minimize]LPS increased the translocation of p50 subunit from the cytosol to the nucleus by 230.7 ± 42.2% (P < 0.05) compared with unstimulated cells. In contrast there was only a small increase in LPS-induced NF-κB activation in the presence of the specific inhibitor of the p50 subunit SN 50 (100 μM) by 88.6 ± 13.6% (NS). PD 098059 (10 μM), SB 203580 (10 μM), and TSA (100 μM) had no effect of the p50 translocation of NF-κB (data not shown).
Consistent with previous studies on macrophages and monocytes (21) in unstimulated cells, an NF-κB:DNA complex was always detectable. LPS increased NF-κB:DNA binding in a concentration- and time-dependent manner with maximum effects at 10 ng/ml and 60 min, respectively (Figure 3A)
Figure 3. (A) Representative EMSA autoradiogram showing the time-course of LPS-induced NF-κB:DNA binding. LPS (10 ng/ml) induced time-dependent increase in NF-κB:DNA binding with a maximum effect at 60 min. (B) Representative EMSA autoradiogram demonstrating the lack of effect of PD 098059 (10 μM) and SB 203580 (10 μM) of NF-κB:DNA binding. However, SN 50 (100 μM) completely inhibited the binding. Supershift analysis was performed using antisera raised against the amino-terminus of the nuclear translocation sequence of p50 subunit of NF-κB.
[More] [Minimize]The effects of inhibition of p50 translocation by SN 50, of IKK-2-inhibition by AS602868 and also of inhibition of phosphorylation of MAP kinases by PD 098059 and SB 203580 on HAT activity were examined. LPS (10 ng/ml) increased HAT activity by 165.8 ± 11.1% compared with basal values (P < 0.01). PD 098059 suppressed LPS-induced HAT activity by ∼ 83%, in contrast to SB 203580, which only inhibited by ∼ 9%, indicating that mainly MKK-1 phosphorylation modulates the regulation of HAT activity. SN 50 decreased LPS-induced HAT activation by ∼ 93% and AS602868 by 66% (10−5 M), indicating that NF-κB is involved in the regulation of HAT activity. Interestingly, there is a potentiating effect of inhibition of HAT activity when p38 MAPK is inhibited in the presence of IKK-2 inhibitor AS602868 (∼ 80%, 10−5 M), and to a lesser degree when MKK-1 is inhibited in the presence of AS602868 (∼ 74%, 10−5 M) (Figure 4)
Figure 4. LPS-induced increase in HAT activation alone and in the presence of PD 098059 (10 μM), SB 203580 (10 μM), SN 50 (100 μM), and TSA (10 ng/ml) measured by nonradioactive HAT indirect ELISA assay. Data shown as mean ± SEM of four determinations, using alveolar macrophages from different donors. **P < 0.01 compared with LPS; §P < 0.05 compared with SB 203580 and LPS.
[More] [Minimize]The effects of post-transcriptional modifications such as phosphorylation of HAT on HAT activity was examined. Basal HAT activity in alveolar macrophages was 4,044 ± 434 dpm (n = 5). LPS (10 ng/ml) induced an increase in HAT activity, as measured by 3H-acetate incorporation into histones, by 129.3 ± 75.9% compared with control (P < 0.05). PD 098059 decreased LPS-induced HAT-activity by 30.1 ± 7.2%, whereas inhibition of HAT activity by SB 203580 was not significantly different (23.1 ± 16.6%) (data not shown).
The specific inhibitor of histone deacetylase (HDAC) Trichostatin A (10 ng/ml) increased LPS-induced GM-CSF release by 15 ± 3.3% compared with LPS alone. PD 098059 did not affect the additional LPS-induced increase in GM-CSF release in the presence of TSA compared with LPS-induced GM-CSF release without TSA. However, in the presence of the specific p38MAPK inhibitor SB 202589 there was an additional increase in LPS-induced GM-CSF release by 33 ± 23.3% when TSA was added compared with SB 203580 alone (P < 0.05; Figure 5)
Figure 5. Additive inhibition of HDAC by trichostatin A (10 ng/ml) on LPS-mediated GM-CSF release in the presence or absence of PD 098059 (10 μM), SB 203580 (10 μM), SN 50 (100 μM), or AS602868 (10−5 M or 10−6 M). Results are presented as mean ± SEM of at least 4–6 independent experiments, using alveolar macrophages from different donors; *P < 0.05 compared with absence of TSA.
[More] [Minimize]We demonstrate that in human alveolar macrophages, GM-CSF generation induced by LPS is mainly regulated by the transcription factor, NF-κB. The specificity of LPS-induced activation of NF-κB in AM was confirmed by supershifting the NF-κB:DNA binding complex in the presence of the antibody to the p50 subunit of NF-κB. Furthermore by the effect of SN 50, a cell-permeable peptide and specific translocation-inhibitor of the p50 subunit of NF-κB in suppressing the LPS-induced translocation of p50. SN50 also prevented the LPS-induced NF-κB:DNA binding in electromobility shift assays and inhibited GM-CSF release. This is in contrast to previous findings where SN 50 had no effect on LPS-induced GM-CSF release in human monocytes (12), indicating a difference in gene regulation during differentiation of monocytes to macrophages, a process which is associated with a loss of transcription factors, REF-1 and AP-1 (22). The specificity of this effect was confirmed by the finding that the inhibition of the protein kinase IκB-kinase-2 (IKK-2) by AS602868 also prevented GM-CSF release in a concentration-dependent manner. Therefore, the generation of GM-CSF in human AM induced by LPS is primarily regulated at the level of gene transcription by NF-κB.
We next investigated the role for phosphorylation and activation of extracellular signal–regulated kinases p44ERK1, p42ERK2, and p38MAPK in GM-CSF generation by studying the ability of their respective inhibitors PD 098059 and SB 203580 to suppress the generation and expression of GM-CSF. PD 098059 decreased GM-CSF release by 77% but only reduced steady-state mRNA by 50% at a concentration that abolished ERK-1 and ERK-2 phosphorylation; it did not affect NF-κB p50 translocation, NF-κB:DNA binding and HDAC inhibition. Therefore, the ERK pathway regulates GM-CSF expression at both the transcriptional and post-transcriptional levels. ERK-1 and -2 may regulate GM-CSF release either downstream of DNA binding in the NF-κB cascade, by an NF-κB pathway independent of NF-κB:DNA binding (10), or by an NF-κB–independent effect on GM-CSF transcription or translation. In contrast, SB 203580, an inhibitor of the p38 MAPK pathway, inhibited GM-CSF release only by ∼ 20% but mRNA synthesis by 50% at a concentration that completely suppressed p38 MAP kinase phosphorylation and with no effect on NF-κB:DNA binding and p50 translocation in LPS-stimulated macrophages. This suggests a different role for p38 MAP kinase pathway in the transcriptional and post-transcriptional regulation of GM-CSF in alveolar macrophages.
The results are consistent with those of Meja and coworkers (12), who reported that PD 098059 (10 μM) inhibits LPS-induced GM-CSF generation in human monocytes by ∼ 73%. In contrast, SB 203580 reduced LPS-stimulated GM-CSF generation in monocytes by ∼ 79% compared with ∼ 20% in our study.
Chromatin is a highly organized and dynamic protein-DNA complex and in resting cells, DNA is compacted to prevent transcription factor accessibility. During activation of the cell, this compactness of DNA is reduced, allowing DNA-binding proteins to bind to DNA, leading to the induction of gene transcription (16). The basal subunit of chromatin, the nucleosome, is composed of an octamer of 4 core histones; an H3/H4 tetramer and two H2A/H2B dimers, surrounded by 146 bp of DNA (23). Specific lysine residues in the N-terminal tails of the core histone can be post-translationally modified by acetylation of the ε-amino group. The dynamic equilibrium of core histone acetylation is established and maintained by HAT and HDAC activities (20). Several transcriptional regulators such as CBP and pCAF possess intrinsic HAT activities, suggesting that histone acetylation play an important role in regulating gene transcription (24–27). Hypoacetylation induced by HDACs leads to a more compact nucleosomal structure and is associated with repression of gene induction (23). Thus, analysis of the HAT activity of a cell reflects its activation status and results from a combination of HAT and HDAC activities within the cell.
We have shown for the first time that LPS induces an increase in HAT activity and that this was suppressed by PD 098059 but only slightly when SB 203580 was added, indicating that ERK-1 and ERK-2 can modify HAT activity post-transcriptionally. Additionally, HAT activity was decreased by inhibition of IKK-2 and also by SN 50. The inhibition of HAT activity was obtained when SB 203580 was combined with the IKK-2 inhibitor, AS602868, indicating that p38 MAPK modulates HAT activity indirectly by modifying the NF-κB pathway rather through a direct effect on HAT activity. There was an additional increase in LPS-induced GM-CSF release when the specific inhibitor of HDAC, trichostatin A, was added, indicating that post-transcriptional inhibition of histone deacetylation can modulate GM-CSF release. This effect of TSA was increased by SB 203580 with an additional increase in LPS-induced GM-CSF release.
In conclusion, we have shown that in human macrophages LPS induces GM-CSF release through an NF-κB–mediated pathway which can be modulated by the MAP kinase pathways, p38 MAPK and ERK-1, -2 acting either transcriptionally or both transcriptionally and post-transcriptionally respectively on NF-κB activation and GM-CSF release. The capacity of inhibitors to modify LPS-induced histone acetylation also suggests that this plays a role in NF-κB–mediated LPS-induced GM-CSF release.
Andrea Koch was supported by the Deutsche Akademie der Naturforscher Leopoldina, Halle, Germany (BMBF-LPD 9701-12); by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany (KO-1788/3-1); by the Lise-Meitner- Habilitations-Program of the Ministerium für Schule, Wissenschaft und Forschung des Landes Nordrhein Westfalen (44-6037.5), Germany; and by the Köln Fortune Program, Faculty of Medicine, University of Cologne (project 8/2003).
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