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• W2038871743 abstract "The lung innate immune response to lipopolysaccharide (LPS) coordinates cellular inflammation, mediator, and protease release essential for host defense but deleterious in asthma, chronic obstructive pulmonary disease, and cystic fibrosis. In vitro, LPS signals to the transcription factors NFκB via TLR4, MyD88, and IL-1R-associated kinase (IRAK), to AP-1 by mitogen-activated protein (MAP) kinases, and via an alternate route in IRAK-deficient mice, but the in vivolung signaling pathway(s) are not understood. We investigated the role of Akt and Erk1/2 as LPS intensely stimulates granulocyte/macrophage-colony-stimulating factor (GM-CSF) release, and neutralizing GM-CSF profoundly suppressed LPS-induced inflammation, suppressed expression and activity of lung proteases, significantly reduced GM-CSF and tumor necrosis factor α (TNFα) mRNA expression, and dampened nuclear localization of both NFκB (p50/65) and AP-1. LPS markedly activated Akt and Erk1/2, but not p38, in a GM-CSF-dependent manner in direct temporal association with NFκB and AP-1 activation. Pharmacological inhibition of Akt or Erk activation in LPS-treated tracheal explants ex vivoinhibited the release of GM-CSF. These data implicate GM-CSF-dependent activation of Akt in the amplification of this response and demonstrate the role of Erks rather than p38 in lung LPS inflammatory responses. Inhibition of GM-CSF may be of therapeutic benefit in inflammatory diseases in which LPS contributes to lung damage. The lung innate immune response to lipopolysaccharide (LPS) coordinates cellular inflammation, mediator, and protease release essential for host defense but deleterious in asthma, chronic obstructive pulmonary disease, and cystic fibrosis. In vitro, LPS signals to the transcription factors NFκB via TLR4, MyD88, and IL-1R-associated kinase (IRAK), to AP-1 by mitogen-activated protein (MAP) kinases, and via an alternate route in IRAK-deficient mice, but the in vivolung signaling pathway(s) are not understood. We investigated the role of Akt and Erk1/2 as LPS intensely stimulates granulocyte/macrophage-colony-stimulating factor (GM-CSF) release, and neutralizing GM-CSF profoundly suppressed LPS-induced inflammation, suppressed expression and activity of lung proteases, significantly reduced GM-CSF and tumor necrosis factor α (TNFα) mRNA expression, and dampened nuclear localization of both NFκB (p50/65) and AP-1. LPS markedly activated Akt and Erk1/2, but not p38, in a GM-CSF-dependent manner in direct temporal association with NFκB and AP-1 activation. Pharmacological inhibition of Akt or Erk activation in LPS-treated tracheal explants ex vivoinhibited the release of GM-CSF. These data implicate GM-CSF-dependent activation of Akt in the amplification of this response and demonstrate the role of Erks rather than p38 in lung LPS inflammatory responses. Inhibition of GM-CSF may be of therapeutic benefit in inflammatory diseases in which LPS contributes to lung damage. lipopolysaccharide granulocyte/macrophage-colony-stimulating factor extracellular signal-regulated kinase mitogen-activated protein MAP kinase IL-1R-associated kinase tumor necrosis factor Toll-like receptor 4 monoclonal antibody c-Jun NH2-terminal kinase phosphate-buffered saline Bronchoalveolar lavage BAL fluid enzyme-linked immunosorbent assay analysis of variance matrix metalloprotease MAP/ERK kinase MEK kinase recombinant The immediate recognition of bacteria and their products in the lung is mediated by an ancient immune response that utilizes conserved pattern recognition receptors to distinguish pathogen-associated molecular pattern signatures of microbes (1Medzhitov R. Janeway Jr., C.A. Cell. 1997; 91: 295-298Abstract Full Text Full Text PDF PubMed Scopus (1940) Google Scholar). LPS1 is a component of the Gram-negative bacterial cell wall and a potent endotoxin capable of activating innate immunity (2Martin T.R. Am. J. Respir. Cell Mol. Biol. 2000; 23: 128-132Crossref PubMed Scopus (113) Google Scholar). The subsequent inflammatory defense reaction coordinates neutrophil recruitment and macrophage activation through release of TNFα, chemokines, oxygen radical products, and proteases. Although this LPS response is essential for host defense, LPS is also implicated in several human lung diseases, notably cystic fibrosis, chronic obstructive pulmonary disease, and asthma (3Li J.D. Dohrman A.F. Gallup M. Miyata S. Gum J.R. Kim Y.S. Nadel J.A. Prince A. Basbaum C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 967-972Crossref PubMed Scopus (253) Google Scholar, 4Vernooy J.H. Dentener M.A. van Suylen R.J. Buurman W.A. Wouters E.F. Am. J. Respir. Cell Mol. Biol. 2002; 26: 152-159Crossref PubMed Scopus (197) Google Scholar, 5Michel O. Kips J. Duchateau J. Vertongen F. Robert L. Collet H. Pauwels R. Sergysels R. Am. J. Respir. Crit. Care Med. 1996; 154: 1641-1646Crossref PubMed Scopus (519) Google Scholar, 6O'Grady N.P. Preas H.L. Pugin J. Fiuza C. Tropea M. Reda D. Banks S.M. Suffredini A.F. Am. J. Respir. Crit. Care Med. 2001; 163: 1591-1598Crossref PubMed Scopus (189) Google Scholar). LPS binds to soluble LPS-binding protein present in the alveolar fluid and is then transferred to CD14 expressed on alveolar macrophages and bronchial epithelial cells (2Martin T.R. Am. J. Respir. Cell Mol. Biol. 2000; 23: 128-132Crossref PubMed Scopus (113) Google Scholar) and, in conjunction with MD-2 signals via, at least, Toll-like receptor 4 (TLR4). TLR4 initiates an IL-1β receptor-like signaling (7Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4344) Google Scholar) promoting NFκB activation. Currently, this is thought to involve the myeloid differentiation protein (MyD88) and MyD88-adapter-like (Mal) adapter molecules, recruitment and activation of IL-1R-associated kinase (IRAK), and complexing with tumor necrosis factor receptor-associated factor 6 (TRAF6), which activates NFκB through activation of NFκB-inducing kinase (NIK) as reviewed in Refs. 8Bowie A. O'Neill L.A. J. Leukocyte Biol. 2000; 67: 508-514Crossref PubMed Scopus (387) Google Scholar and 9Beutler B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (635) Google Scholar. NFκB subsequently promotes the release of inflammatory cytokines such as TNFα, IL-1β, neutrophil-recruiting chemokines, and matrix metalloproteases. However, mice lacking theIRAK gene remain partially responsive to IL-1 and LPS (10Swantek J.L. Tsen M.F. Cobb M.H. Thomas J.A. J. Immunol. 2000; 164: 4301-4306Crossref PubMed Scopus (233) Google Scholar), suggesting that TLR4 signals via IRAK-dependent and -independent pathways to promote NFκB-mediated cytokine expression. GM-CSF, which was originally purified and cloned from lung tissue of endotoxin-treated mice, is a major survival and proliferative factor for hematopoietic cells and primes mature macrophages, eosinophils, neutrophils, and respiratory epithelium for inflammatory effector functions (11Klein J.B. Buridi A. Coxon P.Y. Rane M.J. Manning T. Kettritz R. McLeish K.R. Cell. Signal. 2001; 13: 335-343Crossref PubMed Scopus (85) Google Scholar). In contrast to almost all other tissues, low level basal GM-CSF is required for normal pulmonary physiology as mice deficient in GM-CSF develop fatal alveolar proteinosis that can be rescued by overexpression of the PU.1 transcription factor (12Shibata Y. Berclaz P.Y. Chroneos Z.C. Yoshida M. Whitsett J.A. Trapnell B.C. Immunity. 2001; 15: 557-567Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Since endogenous GM-CSF regulates the intensity of inflammation and strongly activates Akt and Erk kinases, we reasoned that these GM-CSF-regulated kinases might contribute to the lung LPS response (13Dijkers P.F. van Dijk T.B. de Groot R.P. Raaijmakers J.A. Lammers J.W. Koenderman L. Coffer P.J. Oncogene. 1999; 18: 3334-3342Crossref PubMed Scopus (40) Google Scholar, 14Klein J.B. Rane M.J. Scherzer J.A. Coxon P.Y. Kettritz R. Mathiesen J.M. Buridi A. McLeish K.R. J. Immunol. 2000; 164: 4286-4291Crossref PubMed Scopus (245) Google Scholar). In particular, Akt promotes NFκB activity by phosphorylating IκB kinase-α (IKK-α) in response to TNFα and platelet-derived growth factor (15Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1866) Google Scholar, 16Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1654) Google Scholar), which liberates the transcription factor from IκB to initiate nuclear translocation and DNA binding. In addition, Akt induces trans-activation of the p65/RelA subunit of NFκB in response to oncogenic Ras and IL-1β (17Madrid L.V. Wang C.Y. Guttridge D.C. Schottelius A.J. Baldwin Jr., A.S. Mayo M.W. Mol. Cell. Biol. 2000; 20: 1626-1638Crossref PubMed Scopus (578) Google Scholar), a process required for full activation that is distinct from nuclear translocation and DNA binding. GM-CSF also promotes activation of Erk1/2 MAPK homolog, which belongs to a family of universal signal transduction molecules involved in a wide variety of biological responses including regulation of the AP-1 transcription factor complex. The MAPK homologs (Erk1/2, p38, and JNK) promote AP-1 expression and activity by direct phosphorylation. Since MAPK homologs display overlapping substrate specificities that respond in a cell type- and stimuli-specific manner (18Leppa S. Saffrich R. Ansorge W. Bohmann D. EMBO J. 1998; 17: 4404-4413Crossref PubMed Scopus (289) Google Scholar), we investigated the growth factor-regulated Erk1/2 and stress-activated p38 homologs. In the present study, we performed detailed kinetic analyses of the LPS response and report the in vivo activation of Akt and Erk1/2 kinases but not p38 in LPS-inflamed lungs, which is GM-CSF-dependent. Furthermore, the activation status of Akt and Erk1/2 temporally correlated with NFκB and AP-1 DNA binding activity, implicating the kinases as central signal transduction modules in LPS-induced lung inflammation. Our data indicate the molecular mechanisms by which GM-CSF augments innate immunity to Gram-negative pathogens but also suggest that anti-GM-CSF-mediated suppression of Akt and Erk may have clinical implications in inflammatory diseases characterized by exuberant cytokine and protease expression, such as chronic obstructive pulmonary disease, cystic fibrosis, and asthma, in which LPS has been implicated. Specific pathogen-free male BALB/c mice aged 6–7 weeks old and weighing ∼20 g were obtained from the Animal Resource Center Pty. Ltd. (Perth, Australia), housed at 20 °C on a 12-h day/night cycle in sterile micro-isolators, and fed a standard sterile diet of Purina mouse chow with water allowed ad libitum. All animal handling and experimental procedures, which were performed aseptically, were approved by the Animal Experimental Ethics Committee of the University of Melbourne and conformed to International standards of animal welfare as specified in the National Health and Medical Research Committee (NHMRC) of Australia guidelines. GM-CSF-deficient mice (GM-CSF−/−) and wild-type litter mate controls (GM-CSF+/+) were the generous gift of Professor Ashley Dunn, Ludwig Institute for Cancer Research, Melbourne, Australia (19Stanley E. Lieschke G.J. Grail D. Metcalf D. Hodgson G. Gall J.A. Maher D.W. Cebon J. Sinickas V. Dunn A.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5592-5596Crossref PubMed Scopus (700) Google Scholar). Inflammation was induced by transnasally instilling a maximally tolerated dose of LPS (10 μg of Escherichia ColiSerotype 026:B6, Sigma, in 35 μl of in PBS vehicle) into the lungs of groups of 8–10 mice anesthetized with 2% enflurane (Abbott) in air, which uniformly distributes LPS throughout the lungs (20Blyth D.I. Pedrick M.S. Savage T.J. Bright H. Beesley J.E. Sanjar S. Am. J. Respir. Cell Mol. Biol. 1998; 19: 38-54Crossref PubMed Scopus (81) Google Scholar). Solutions administered to the mice, alone or in combinations at specified time points, were (a) PBS; (b) PBS containing isotype control (rat anti-mouse IgG2a mAb of irrelevant specificity, 100 μg/mouse); (c) PBS containing anti-GM-CSF mAb (22E9, rat anti-mouse IgG2a ultrapurified in-house, 100 μg/mouse); (d) PBS containing LPS. Anti-GM-CSF, PBS, or isotype control was administered 3 h prior to LPS challenge. All reagents were LPS-free and negative in the limulus lysate assay. For necroscopy, mice were anesthetized with ketamine/xylazine (15 mg/kg and 30 mg/kg, intraperitoneally, respectively). Bronchoalveolar lavage (BAL) (four pooled aliquots of 0.3 ml of PBS) recoveries (85 ± 5%) did not differ significantly between groups. In the GM-CSF−/− mice only, which suffer from alveolar proteinosis (19Stanley E. Lieschke G.J. Grail D. Metcalf D. Hodgson G. Gall J.A. Maher D.W. Cebon J. Sinickas V. Dunn A.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5592-5596Crossref PubMed Scopus (700) Google Scholar), BAL samples were centrifuged over a 15% BSA in PBS (with 0.5 m EDTA) density gradient. Total cell counts and viabilities were determined by ethidium bromide/acridine orange (Molecular Probes) fluorescent viability stains using Neubauer hemocytometer. Cytocentrifuge preparations (Shandon Cytospin 3) using 100 μl of BAL were differentiated according to standard morphological criteria counting at least 500 cells/(DiffQuik, Ziess, Axiolab, ×1000). Murine GM-CSF and TNFα levels were analyzed according to the manufacturer's instructions (Pharmingen, limit of detection 10 ng/ml). Following lavage, the lungs were perfused via the right ventricle with 5 ml of PBS to remove intravascular leukocytes, removed, snap-frozen in liquid nitrogen, ground, and stored at −80 °C. For preparation of whole cell extracts, 10 mg of ground lung tissue was resuspended in 500 μl of lysis buffer (50 mmTris-HCl (pH 7.5), 120 mm NaCl, 1% (v/v) Nonident P-40, 1 mm EDTA, 50 mm NaF, 40 mmβ-glycerophosphate, 1 mm benzamadine, and 0.5 mm phenylmethylsulfonyl fluoride). Following a 15-min incubation on ice, homogenates were cleared by centrifugation for 10 min (16000 × g at 4 °C). For nuclear extraction, 10 mg of lung tissue was resuspended in 500 μl of nuclear lysis buffer 1 (10 mm HEPES (pH 7.6), 15 mm KCl, 2 mm MgCl2, 0.1 mm EDTA, 5 mm β-mercaptoethanol, 0.2% Nonident P-40, and 0.5 mm phenylmethylsulfonyl fluoride) for 10 min on ice. Nuclei were pelleted by centrifugation at 800 × g for 30 s and lysed in 500 μl of nuclear lysis buffer 2 (50 mmHEPES (pH 7.6), 400 mm KCl, 0.1 mm EDTA, 10% glycerol, 5 mm β-mercaptoethanol, and 0.5 mmphenylmethylsulfonyl fluoride). Following a 30-min incubation on ice, the nuclei extract (supernatant) was retained following centrifugation for 10 min (800 × g at 4 °C). Protein concentrations from whole and nuclear extracts were determined using the Dc protein assay (Bio-Rad), and all extracts were stored at −80 °C. Total RNA was isolated from 10 mg of whole lung tissue according to the manufacturers' instructions using the Rneasy kit (Qiagen). The purified total RNA prep was used as a template to generate cDNA. The reaction mix containing 1 μg of RNA, 0.5 μg of random hexamers (Promega), 15 units of avian myeloblastosis virus reverse transcriptase enzyme (Promega), and 20 units of Rnasin (Promega) in reverse transcriptase buffer (50 mm Tris-HCl (pH 8.3), 50 mm KCl, 10 mm MgCl2, 10 mm dithiothreitol, and 0.5 mm spermadine) was incubated at 37 °C for 60 min. The reaction was terminated at 90 °C for 2 min and cooled to 4 °C. The quantitative PCR was performed by ABI PRISM 7900HT sequence detection system (Applied Biosystems) using predeveloped Taqman probe/primer combinations for GM-CSF, 18 S rRNA, and TNFα optimized by the manufacturer. Taqman PCR was performed in 10-μl volumes using AmpliTaq Gold polymerase and universal reaction buffer (Applied Biosystems). Threshold cycle numbers were transformed using the ΔΔCt and relative value method as described by the manufacturer and were expressed relative to 18 S rRNA, which was used as a housekeeping gene by multiplexing single reactions. The data were then compared with levels in the PBS control group and are presented as fold increase over PBS alone. The following pairs of complimentary oligonucleotides with 4 nucleotide overhangs were used to generate double-stranded DNA for NFκB and AP1 binding sites: NFκB sense, 5′-CATGCAACAGAGGGGACTTTCCGAGAGG and NFκB antisense, 5′-CATGCCTCTCGGAAAGTCCCCTCTGTTG-3′; AP1 sense, 5′-CATGCGCTTGATGAGTCAGCCGGAA-3′ and AP1 antisense, 5′-CTAGTTCCGGCTGACTCATCAAGCG-3′. Complimentary pairs of oligonucleotides (50 ng) were annealed and radiolabelled using 5 units of Klenow fragment (Promega) in the presence of 50 mmTris-HCl (pH 7.2), 10 mm MgSO4, 0.1 mm DTT, 0.2 mm dGTP, 0.2 mm dCTP, 0.2 mm dTTP, and 100 μCi of [α-32P]dATP (Geneworks). Labeled probes were then separated from unincorporated isotope by size exclusion chromatography using Microspin G-25 columns (Amersham Biosciences). Nuclear extracts (5 μg) were used in binding assays in 20-μl reactions and incubated at room temperature with labeled probe (50,000 cpm) in the presence of 10 mmTris-HCl (pH 7.5), 0.05 mg/ml poly[dI-dC]·poly[dI-dC], 4% glycerol, 1 mm MgCl2, 0.5 mmdithiothreitol, and 50 mm NaCl for 20 min. Following incubation, 15 μl of binding reaction was immediately resolved on a 7.5% gel slab (37.5 acrylamide: 1 bis-acrylamide) in 0.5× TBE at 200 V for 20–30 min. Gels were prepared 1 day prior to experiment and prerun for 30 min before binding reactions were loaded. Gels were then dried and exposed to autoradiography using an Intensifier screen at −80 °C. Densitometry was performed using Kodak EDAS 1D image analysis software. The specificity of the assay was confirmed in separate shift assays on the same nuclear extracts, demonstrating that antibody to the p50 subunit of NFκB supershifted both bands (not shown). Whole cell extracts (30 μg) were diluted in Laemmli sample buffer, denatured at 90 °C for 5 min, and subjected to SDS-PAGE on 10% gel slabs with 5% stacking gels at 200 V for 45 min. Resolved proteins were transferred onto Hybond polyvinylidene difluoride membrane (Amersham Biosciences) using Trans-blot S.D. transfer cell (Bio-Rad) at 10 V for 30 min. Following transfer, polyvinylidene difluoride membranes were incubated in blocking solution (Tris-buffered saline containing 5% (w/v) skim milk powder and 0.5% (v/v) Tween 20) for 1 h at room temperature. The activation status of Akt, Erk1/2, and p38 was then assessed using phospho-specific antibodies (Cell Signaling, Beverly, MA), which recognize activated forms of these kinases. Actin levels were also assessed as a loading control using an antibody (Santa Cruz Biotechnology, Santa Cruz, CA) that reacts with a broad range of actin isoforms. Membranes were incubated with primary antibodies diluted in blocking solution overnight at 4 °C. Following primary incubation, membranes were washed three times in blocking solution and incubated for 1 h with horseradish peroxidase-conjugated anti-IgG (Bio-Rad). A final wash over 30 min with six changes in wash buffer (Tris-buffered saline containing 0.5% Tween 20) was performed. Immunoreactive bands were finally visualized by autoradiography using chemiluminescence (ECL, Amersham Biosciences) with exposure times of 30 s to 2 min. Densitometry was performed using Kodak EDAS 1D image analysis software. Zymography was used to assess protease expression in response to LPS treatment. Briefly, SDS-PAGE mini-gels (10%) were prepared with the incorporation of gelatin (2 mg/ml, Labchem) before casting. BALF (10 μl) was run into gels at a constant voltage of 200 V under non-reducing conditions. When the dye front reached the bottom, gels were removed and washed twice for 15 min in 2.5% Triton X-100 and incubated at 37 °C overnight in zymography buffer (50 mm Tris-HCl (pH 7.5), 5 mm CaCl2, 1 mm ZnCl2and 0.01% NaN3). The gels were then stained for 45 min with Coomassie Brilliant Blue R-250 (Sigma) and extensively destained. Following destaining, zones of enzyme activity appeared clear against the Coomassie Blue background. BALF was also tested for net gelatinase activity using fluorescence-conjugated gelatin (Molecular Probes). The gelatin substrate (10 μg) was diluted in 50 mm Tris pH 7.5, 150 mm NaCl, 5 mm CaCl2, 0.01% NaN3 and incubated at room temperature for 16 h with 100 μl of neat BALF. The digested substrate has absorption/emission maxima at 495 nm/515 nm, and its fluorescence intensity was measured in a microplate reader (Victor II, Wallac) to detect quantitative differences in activity. Trachea and main bronchi were isolated free of adherent connective tissue from untreated anesthetized mice and placed in a 1.5-ml Eppendorf tube containing 250 μl of RPMI. For vehicle treatments, 0.1% Me2SO (final v/v) was added. Wortmannin (Sigma) and U0126 (Cell Signaling) were added to a final concentration of 1 and 10 μm, respectively, for 30 min at 37 °C, rotating at 750 rpm in a thermo-mixer. Mouse serum (1%, final v/v) prepared in-house and LPS (1 μg/ml) were then added to individual tubes, and at 0, 15 min and 3 h, the trachea explants were briefly spun down, and the neat supernatant was retained for GM-CSF ELISA. Tracheas were immediately snap-frozen in liquid nitrogen, and protein extracts were prepared as detailed previously using 200 μl of lysis buffer. As all data were normally distributed, responses were analyzed by ANOVA followed by Dunnett's multiple comparison test. Unless otherwise specified, data were reported as mean ± S.E. Group sizes in all experiments were at least 8 based on power calculations made to detect a 15% difference from control forp < 0.05, the level of statistical significance. Note that all experiments were replicated at least three times with identical statistical outcomes. Consistent with previous studies (20Blyth D.I. Pedrick M.S. Savage T.J. Bright H. Beesley J.E. Sanjar S. Am. J. Respir. Cell Mol. Biol. 1998; 19: 38-54Crossref PubMed Scopus (81) Google Scholar), 10 μg of LPS/mouse caused marked neutrophilic inflammation but not lung hemorrhage or acute respiratory distress. Administration of anti-GM-CSF 3 h prior to LPS challenge markedly suppressed LPS-induced neutrophilia (>85%), whereas an equivalent dose of isotype control exerted no significant effect on neutrophil recruitment into the lavage of BALB/c mice (Fig.1 A). The degree of inhibition was equivalent to the suppression of neutrophilia observed in confirmatory experiments in GM-CSF−/− mice (Fig.1 B). Anti-GM-CSF recovered by BAL after instillation completely neutralized recombinant GM-CSF (rGM-CSF) but not rTNFα (Fig. 2 A). Anti-GM-CSF also blocked detection of secreted GM-CSF but not TNFα protein in LPS-challenged mice, which were markedly enhanced in saline- and LPS-treated mice (Fig. 2 B).Figure 2Anti-GM-CSF neutralizes recombinant and endogenous GM-CSF present in BALF and inhibits expression of GM-CSF and TNF α transcript. As shown in A, ELISA was used to measure recombinant GM-CSF and TNFα prepared in the BALF of PBS (▪) or anti-GM-CSF-treated and LPS-exposed animals (▴) over the specified concentration range. B, endogenous secreted GM-CSF and TNFα secreted in BALF of PBS (clear bars) or LPS (black bars, 10 μg for 2 h)-challenged mice pretreated with and without 100 μg of anti-GM-CSF/mouse. #, p < 0.05; ANOVA/Dunnett's test, significantly different from saline and LPS group (n = 4). As shown in C, total RNA was isolated from PBS (clear bar) and LPS-challenged lungs (black bars, 10 μg for 2 h) pretreated in the absence or presence of anti-GM-CSF. RNA was reverse-transcribed using random hexamers and subjected to Taqman PCR to detect GM-CSF and TNFα transcript relative to 18 S rRNA by multiplexing single reactions. The data were then compared with levels in the PBS control group and are presented as fold increase over PBS alone. #, p < 0.05; ANOVA/Dunnett's test, significantly different from saline and LPS group (n = 4).View Large Image Figure ViewerDownload (PPT) To establish whether anti-GM-CSF exerted its inhibitory effect at the transcript level, real time fluorescence (Taqman) PCR was used to assess GM-CSF and TNFα transcript expression normalized to 18 S rRNA. Consistent with in vitro models (21Meja K.K. Seldon P.M. Nasuhara Y. Ito K. Barnes P.J. Lindsay M.A. Giembycz M.A. Br. J. Pharmacol. 2000; 131: 1143-1153Crossref PubMed Scopus (71) Google Scholar), LPS promoted expression of GM-CSF and TNFα mRNA in vivo (Fig.2 C). Neutralizing GM-CSF significantly inhibited expression of both GM-CSF and TNFα mRNA by ∼26 and 44% respectively, suggesting that GM-CSF may play a role in priming or promoting activation of the inflammatory transcriptional machinery responsible for coordinating gene expression. The discrepancy between TNFα protein and transcript implies that preformed TNFα stores may be secreted in an anti-GM-CSF-resistant manner and are consistent with previous studies demonstrating that protein synthesis inhibition dampens but does not completely abolish LPS-induced TNFα release in the lung in vivo (22Goncalves de Moraes V.L. Boris Vargaftig B. Lefort J. Meager A. Chignard M. Br. J. Pharmacol. 1996; 117: 1792-1796Crossref PubMed Scopus (93) Google Scholar). Since MMPs contribute to the movement of neutrophils into the lung parenchyma and MMP9 expression in response to LPS is transcriptionally regulated by NFκB and AP-1 (23Kim H. Koh G. Biochem. Biophys. Res. Commun. 2000; 269: 401-405Crossref PubMed Scopus (89) Google Scholar, 24Aljada A. Ghanim H. Mohanty P. Hofmeyer D. Tripathy D. Dandona P. J. Clin. Endocrinol. Metab. 2001; 86: 5988-5991Crossref PubMed Scopus (41) Google Scholar), we investigated the secretion of MMP9 in the BALF of LPS-challenged mice by gelatin zymography (Fig.3 A). LPS potently stimulated protease release in the BALF as assessed by clear regions corresponding to zones of degradation. Major bands of protease activity were identified at ∼90 kDa, corresponding to the molecular size of latent MMP9 (92 kDa) and a major band activity band corresponding to the active form of MMP9 (86 kDa). Additionally, higher molecular size protease bands were observed, corresponding to previously described dimers or complexes of MMP9 (25Zheng T. Zhu Z. Wang Z. Homer R.J., Ma, B. Riese Jr., R.J. Chapman Jr., H.A. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (528) Google Scholar). We did not observe bands corresponding to MMP2 (72 kDa) or matrilysin (MMP7, 28 kDa). Anti-GM-CSF reduced the expression of MMP9 by about 50%. Net protease activity in neat lavagates was also assessed using gelatin conjugated to a fluorescence probe. Consistent with the zymography, LPS-induced net gelatinase activity was partially suppressed by prior treatment with anti-GM-CSF (Fig. 3 B). As expression of GM-CSF, TNFα, and MMP9 are under the regulation of multiple transcription factors including NFκB and AP-1 (26Coles L.S. Diamond P. Occhiodoro F. Vadas M.A. Shannon M.F. J. Biol. Chem. 2000; 275: 14482-14493Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 27Jongeneel C.V. Immunobiology. 1995; 193: 210-216Crossref PubMed Scopus (67) Google Scholar), we examined their DNA binding activity by electromobility shift assay using nuclear extracts from LPS-challenged lungs. Their respective activities were assessed in a kinetic study in the absence or presence of neutralizing anti-GM-CSF (Fig.4 A) and presented graphically following densitometry analysis (Fig. 4 B). LPS induced the DNA binding activity of nuclear NFκB and AP-1, and maximal activity was maintained between 1 and 6 h. Importantly, anti-GM-CSF reduced the activity of NFκB by 30–40% during the maximal activation period, consistent with the decline in TNFα transcript and MMP9 expression. The baseline activity of AP-1 was slightly elevated by anti-GM-CSF mAb, possibly due to anti-GM-CSF antibody binding to Fc receptors on lung mast cells or macrophages (28Turner H. Cantrell D.A. J. Exp. Med. 1997; 185: 43-53Crossref PubMed Scopus (61) Google Scholar). Fc receptor signaling is known to feed into the Ras pathway for AP-1 activation (28Turner H. Cantrell D.A. J. Exp. Med. 1997; 185: 43-53Crossref PubMed Scopus (61) Google Scholar), but in the absence of concurrent basal NFκB activation (Fig. 1), this AP-1 response alone is clearly not sufficient to cause neutrophilic inflammation in vivo. This LPS-induced activation of AP-1 was suppressed by anti-GM-CSF, as maximal activation between 1 and 6 h was reduced by 50%. 1L-1β receptor activates NFκB by promoting the dissociation of IκB from NFκB, which is followed by the Akt-mediated trans-activation of p65/RelA for complete activation (7Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4344) Google Scholar). Similarly, TLR4 promotes IκB ubiqitination; however, the role of Akt in LPS-induced lung inflammation remains unclear. Furthermore, the activation of two MAPK homologs, Erk1/2 and p38, is known to be important in AP-1 regulation (18Leppa S. Saffrich R. Ansorge W. Bohmann D. EMBO J. 1998; 17: 4404-4413Crossref PubMed Scopus (289) Google Scholar); however MAPK substrate specificities in our mouse model have not been established. Therefore, we examined Akt, Erk1/2, and p38 activity in whole lung extracts derived from LPS-challenged mice by Western analysis using phospho-specific antibodies that only recognize activated forms of their respective kinases. Kinase activity was assessed in a kinetic study over 24 h, and actin was also immunoblotted as an internal loading control (Fig.5 A). Densitometry analysis was performed, and activities were presented as a fold increase above baseline (Fig. 5 B). LPS rapidly induced activation of Akt and Erk1/2 within 15 min, suggesting that Akt and Erk are functional modules of the TLR4 signaling cas" @default.
• W2038871743 created "2016-06-24" @default.
• W2038871743 creator A5008084743 @default.
• W2038871743 creator A5026903949 @default.
• W2038871743 creator A5049203385 @default.
• W2038871743 creator A5060211723 @default.
• W2038871743 creator A5079475266 @default.
• W2038871743 date "2002-11-01" @default.
• W2038871743 modified "2023-10-18" @default.
• W2038871743 title "Granulocyte/Macrophage-Colony-stimulating Factor (GM-CSF) Regulates Lung Innate Immunity to Lipopolysaccharide through Akt/Erk Activation of NFκB and AP-1 in Vivo" @default.
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