Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 2014 Dec 8;144(1):79–90. doi: 10.1111/imm.12352

S100A8, S100A9 and S100A12 activate airway epithelial cells to produce MUC5AC via extracellular signal-regulated kinase and nuclear factor-κB pathways

Jin Hyun Kang 1, Sae Mi Hwang 2, Il Yup Chung 1,2
PMCID: PMC4264912  PMID: 24975020

Abstract

Airway mucus hyperproduction is a common feature of chronic airway diseases such as severe asthma, chronic obstructive pulmonary disease and cystic fibrosis, which are closely associated with neutrophilic airway inflammation. S100A8, S100A9 and S100A12 are highly abundant proteins released by neutrophils and have been identified as important biomarkers in many inflammatory diseases. Herein, we report a new role for S100A8, S100A9 and S100A12 for producing MUC5AC, a major mucin protein in the respiratory tract. All three S100 proteins induced MUC5AC mRNA and the protein in normal human bronchial epithelial cells as well as NCI-H292 lung carcinoma cells in a dose-dependent manner. A Toll-like receptor 4 (TLR4) inhibitor almost completely abolished MUC5AC expression by all three S100 proteins, while neutralization of the receptor for advanced glycation end-products (RAGE) inhibited only S100A12-mediated production of MUC5AC. The S100 protein-mediated production of MUC5AC was inhibited by the pharmacological agents that block prominent signalling molecules for MUC5AC expression, such as mitogen-activated protein kinases, nuclear factor-κB (NF-κB) and epidermal growth factor receptor. S100A8, S100A9 and S100A12 equally elicited both phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear translocation of NF-κB/degradation of cytosolic IκB with similar kinetics through TLR4. In contrast, S100A12 preferentially activated the ERK pathway rather than the NF-κB pathway through RAGE. Collectively, these data reveal the capacity of these three S100 proteins to induce MUC5AC production in airway epithelial cells, suggesting that they all serve as key mediators linking neutrophil-dominant airway inflammation to mucin hyperproduction.

Keywords: airway epithelial cells, MUC5AC, S100A12, S100A8, S100A9

Introduction

Asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis are prevalent lung diseases associated with mucus hyperproduction and accumulation in airways. Mucus hyperproduction is a key pathophysiological feature of these diseases, as it plugs airways and impairs mucociliary clearance of pathogens and toxic particles, resulting in airway obstruction and airflow limitation. Diseases with a mucus hypersecretory phenotype are often characterized by an abnormal immune response with increased numbers of neutrophils in the lungs from patients with severe asthma,1,2 COPD3 and cystic fibrosis.4 Corticosteroids, the most widely used anti-inflammatory therapy for the treatment of these types of disease, have limited therapeutic benefits, as they incompletely suppress neutrophil function and infiltration.5 Neutrophilic airway inflammation is probably the principal effector of airway damage and remodelling associated with airway obstruction in these severe diseases. Activation and degranulation of lung neutrophils lead to the release of reactive oxygen species, mediators and proteases responsible for tissue damage/repair and innate immune responses.6 Notably, neutrophil elastase (NE) is prominent due to its ability to cause mucin hyperproduction.7,8 There is currently no specific therapy to resolve mucin hypersecretion.

MUC5AC is the predominant mucin in airway mucus and sputum, and is the most specific marker of goblet cells.9 In the lungs, goblet cell hyperplasia/metaplasia of epithelial cells in inflammatory diseases correlates closely with increases in the expression of MUC5AC.10 A number of inflammatory mediators that stimulate MUC5AC gene expression in airway epithelial cells in vitro or in vivo have been identified, including pro-inflammatory cytokines, growth factors, neutrophil and eosinophil products, bacterial and viral products, and chemical agents in the environment.11 These stimuli induce MUC5AC expression through both distinct and overlapping signal pathways. Numerous studies have clearly demonstrated that the nuclear factor-κB (NF-κB)12,13 and extracellular signal-regulated kinase (ERK) pathways14–16 play a major role in mediating the expression of MUC5AC. Further, the proximal promoter of the MUC5AC gene contains multiple putative binding sites for NF-κB and Sp1,17 in which the latter transcription factor is activated by the epidermal growth factor receptor (EGFR) –ERK pathway.14,16

It has become increasingly evident that damage-associated molecular pattern (DAMP) molecules are recognized as newly identified extracellular danger signals that initiate and modulate local inflammation and innate immune responses when they are actively released following cytokine stimulation as well as passively during cell death.18 S100 proteins comprise a group of DAMP molecules considered to be important inflammatory mediators, and are calcium-binding proteins characterized by EF-hand motifs connected by a central hinge region.18 In particular, S100A8, S100A9 and S100A12 are expressed in great abundance by neutrophils, representing 40% of the cytosolic content of granulocytes.19 These three S100 proteins bind to and activate responses by two widely expressed but divergent receptors, namely, Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE).18,20–23 The engagement of these two receptors by S100 proteins is linked to an array of signalling pathways, notably NF-κB and mitogen-activated protein kinases,20–22,24,25 which overlap considerably with the signalling pathways for MUC5AC expression in response to a wide variety of stimuli. Although it is presumed that S100 proteins regulate or directly cause MUC5AC expression, this has not yet been directly investigated.

The S100A8, S100A9 and S100A12 proteins are associated with numerous inflammatory diseases.18 Specifically, they are markedly elevated in the serum of patients with arthritis, systemic autoimmune diseases and chronic inflammatory bowel diseases, and so have been suggested as biomarkers of inflammation. Some S100 proteins have also been implicated in chronic inflammatory airway diseases. S100A12 provokes activation of mast cells leading to the release of histamine and cytokines,26 while transcripts of S100A9 are up-regulated in peripheral blood mononuclear cells during asthma exacerbation.27 Similarly, S100A9 is also elevated in the sputum of neutrophilic inflammation in severe uncontrolled asthma.28 Together, these findings raise the possibility that certain S100 proteins may be associated with increased risk for obstructive pulmonary diseases. However, the role for these specific S100 proteins in the pathogenesis of such diseases is entirely unknown. In the present study, we show that the three S100 proteins S100A8, S100A9 and S100A12 activate airway epithelial cells to produce MUC5AC, and propose that they serve as important mediators linking neutrophilic-dominant airway inflammation to mucin hyperproduction.

Materials and methods

Materials

S100A8, S100A9 and S100A12 proteins were recombinant products from SinoBiologial Inc. (Beijing, China). S100A8 and S100A9 proteins were produced from recombinant insect cells, while S100A12 was produced from recombinant Escherichia coli. The endotoxin content of all three proteins was < 1·0 EU/μg protein as determined by the Limulus amoebocyte lysate method. Blocking anti-S100A8 and anti-S100A9 antibodies were gifted by Dr Philippe Tessier at Laval University, Canada and were previously described.29 A blocking anti-S100A12 antibody was purchased from Abcam (Cambridge, MA). The two NF-κB reporter plasmids NF-κB-Luc (Panomics, Santa Clara, CA) and NF-κB-hrGFP (Agilent Technologies, Santa Clara, CA) were purchased as indicated.

Cell culture for NCI-H292 and human bronchial epithelial cells

NCI-H292 cells, which are human pulmonary muco-epidermoid carcinoma cells, were cultured in RPMI-1640 medium (Welgene, Daegu, Korea) containing 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml), HEPES (25 mm) and sodium pyruvate (1 mm). Normal human bronchial epithelial (NHBE) cells (Cambrex Bio Science, Baltimore, MD) were prepared as previously described.30 The NHBE cells were cultured in bronchial epithelial cell growth medium (Cambrex Clonetics, Walkersville, MD) supplemented with defined growth factors (SingleQuot kit; Cambrex Clonetics). Medium was replaced every 48 hr until cells were 90% confluent. Cells were seeded at 8·25 × 104 cells/insert on Corning Costar Transwell inserts with 0·4-μm pores (Corning Life Sciences, Lowell, MA) and grown in differentiation medium containing a 1 : 1 mixtures of Dulbecco's modified Eagle's medium and bronchial epithelial cell growth medium. Cells were submerged for 7 days in culture, and then the apical medium was removed to establish an air–liquid interface for 14 days. Medium was refreshed three times weekly. The apical surface of cells was rinsed with PBS once weekly to remove accumulated mucus and debris.

Cell stimulation and inhibitor treatment

At 80% confluence, NCI-H292 cells were serum-starved for 4 hr and treated with S100 proteins for induction of MUC5AC expression for the indicated time periods. NHBE cells that were cultured in air–liquid interface were also stimulated with the S100 proteins. Where various inhibitors for signalling pathways were used, cells were pre-treated with the inhibitors for 30 min before S100 protein treatment. The inhibitors consisted of AG1478, an EGFR inhibitor (Calbiochem, La Jolla, CA), TAPI-2, a TACE inhibitor (Merck, Darmstadt, Germany), BAY11-7082, an NF-κB inhibitor (Calbiochem), U0126, an ERK inhibitor (Calbiochem), SB203580, a p38 inhibitor (Tocris, Ballwin, MO), SP600125, a c-Jun N-terminal kinase (JNK) inhibitor (Tocris), TAK-242, a TLR4 inhibitor (InvivoGen, San Diego, CA), and polymyxin B, an endotoxin inhibitor (Sigma-Aldrich, St Louis, MO). To block RAGE-mediated signalling, NCI-H292 cells were pre-treated with an anti-human RAGE antibody (R&D Systems, Minneapolis, MN) or mouse IgG2 (BD PharMingen, San Diego, CA).

Measurement of luciferase activity

We cloned the proximal promoter sequence of the MUC5AC gene spanning 1 kb into the pGL3 vector. NCI-H292 cells were then transfected with the MUC5AC promoter reporter or one of two NF-κB reporter plasmids using a Lipofectamine 2000-based gene transfer method (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. At 24 hr post transfection, cells were treated with S100 proteins (200 ng/ml) for 8 hr or 12 hr for the NF-κB reporter assay and MUC5AC promoter reporter assay, respectively. Luciferase activity was measured using the Dual-Luciferase Reporter System (Promega, Madison, WI), and transfection efficiency was normalized to Renilla luciferase activity. Transcriptional activity was expressed as the fold increase over cells treated with media alone.

RNA isolation, RT-PCR, and quantitative real-time PCR

Total mRNA was extracted from NCI-H292 and NHBE cells using TRI reagent (Molecular Research Center, Cincinnati, OH). First-strand cDNA was synthesized from 2 μg total RNA using SuperScript II RNase Reverse Transcriptase (Invitrogen) in a 20-μl reaction containing Oligo-dT (12–18mer) primer, deoxynucleotide triphosphates (0·5 mm), MgCl2 (2·5 mm), and dithiothreitol (5 mm). Reverse transcription was performed at 42° for 1 hr and followed by heat inactivation at 70° for 15 min. Synthesized cDNA was amplified for 30 cycles with thermostable DNA polymerase using Hot-start PCR premix (Bioneer, Daejeon, Korea). Quantitative real-time PCR was performed in a 20-μl reaction with 0·5 μl cDNA, 0·8 μl of each primer (10 pm), and 10 μl SYBR Green Master Mix (Applied Biosystems, Foster City, CA) using the Applied Biosystems Prism 7900 Sequence Detection System (Applied Biosystems). The PCR conditions were as follows: 2 min at 50° and 10 min at 95°, followed by 40 cycles of 95° for 30 seconds, 60° for 30 seconds and 72° for 30 seconds. The PCR primers were previously described.30 The specificity of amplification was confirmed by melting curve analysis and gel electrophoresis. Relative expression was evaluated using the comparative cycle threshold (2−ΔΔCt) method and expressed as the mean ± SEM.

Western blot analysis

Cells were first lysed in RIPA buffer (50 mm Tris–HCl, 0·1 m NaCl, 0·1% NaN3, 1% Nonidet P-40, 0·25% sodium deoxycholate, 1 mm EDTA, 1 mm Na3VO4, 1 mm NaF and protease inhibitor mixture) for 20 min at 4° and then centrifuged for 10 min at 4°. The supernatant represented the cytosolic fraction. The pellet was resuspended with modified RIPA buffer (0·4 m instead of 0·1 m NaCl) for 20 min at 4° and centrifuged. The supernatant represented the nuclear fraction. The fractions were resolved by SDS–PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat dry milk and probed with either anti-phospho-ERK1/2 (Cell Signaling Technology, Beverly, MA), anti-phospho-p38 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-JNK (Santa Cruz Biotechnology), anti-phospho-EGFR (12A3: Santa Cruz Biotechnology), anti-p65 NF-κB (Santa Cruz Biotechnology), or anti-IκB (Cell Signaling Technology) antibodies. Anti-ERK2 (C-14: Santa Cruz Biotechnology), anti-p38 (Cell Signaling Technology), anti-JNK (Cell Signaling Technology), anti-EGFR (13G8: Santa Cruz Biotechnology), anti-GAPDH (Santa Cruz Biotechnology), and anti-Histone3 (Abcam) antibodies were used as loading controls. Immunostained proteins were visualized with an ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ).

Immunocytochemistry and immunofluorescent staining

Immunocytochemistry was carried out as previously described.30 For immunofluorescent staining, NCI-H292 cells were fixed in 4% paraformaldehyde, permeabilized in Tris-buffered saline containing 0·1% saponin for 15 min, and incubated at 4° with an anti-MUC5AC antibody (Chemicon, Temecula, CA) conjugated with Quantum dot 605 (Quantum Dot, Hayward, CA). Fluorescence images of immunostained samples were obtained with a Leica TCS-SL confocal microscope (Leica, Heidelberg, Germany).

Statistical analysis

All data were analysed by independent t-tests or analysis of variance using SPSS software. Differences with a P-value < 0·05 were considered statistically significant. Results are expressed as the mean ± standard error of the mean (SEM).

Results

S100A8, S100A9 and S100A12 induce MUC5AC production in airway epithelial cells

We set out to examine whether the three S100 proteins, S100A8, S100A9 and S100A12, induce MUC5AC production in NCI-H292 cells, a human lung carcinoma cell line. When NCI-H292 cells were treated with varying concentrations of the three recombinant human S100 proteins, all three S100 proteins induced MUC5AC mRNA in a dose-dependent manner as determined by real-time PCR. Each of the three S100 proteins had a maximal effect on expression of MUC5AC mRNA at a concentration of 200 ng/ml (Fig. 1a), and MUC5AC mRNA expression reached a maximum after stimulation for 8–12 hr (Fig. 1b). MUC5AC protein was abundantly produced in the cytosolic compartment upon exposure to S100 proteins as determined by immunocytochemistry and immunofluorescent staining (Fig. 1c). Specifically, in parallel with the expression pattern of MUC5AC mRNA, MUC5AC protein expression was up-regulated in a dose-dependent manner, with nearly 30% of the cells exhibiting MUC5AC-positive staining (Fig. 1d). Overall, the levels of MUC5AC expression by these three S100 proteins were comparable to those by EGF (Fig. 1a, d). Furthermore, the three S100 proteins caused a significantly elevated secretion of MUC5AC (see Supporting information, Fig. S1). We cloned the proximal promoter sequence of MUC5AC gene spanning 1 kb, which is known to contain functional binding elements for transcription factors such as NF-κB and Sp1.14,16,17,31 These three S100 proteins weakly but significantly transactivated the MUC5AC promoter by an approximately 1·6-fold increase (Fig. 1e). To evaluate the tendency for the S100 proteins to induce MUC5AC expression in a more physiological setting, NHBE cells were prepared using air–liquid interface culture and stimulated with S100A8, S100A9 and S100A12. The three S100 proteins induced MUC5AC mRNA in a dose-dependent manner (Fig. 1f). Further, the number of MUC5AC-positive cells was clearly increased by treatment with S100A8 as determined by immunofluorescent staining (see Supporting information, Fig. S2). Collectively, these data demonstrated that all three S100 proteins, S100A8, S100A9 and S100A12, activate airway epithelial cells to induce MUC5AC production. To exclude the possibility that the observed effects were due to endotoxin contamination of the recombinant human S100 protein preparations, NCI-H292 cells were treated with the S100 proteins in the presence of polymyxin B, an endotoxin inhibitor. The addition of polymyxin B did not affect MUC5AC mRNA and protein expression at 10 μg/ml (Fig. 2a, b) and 1 μg/ml (data not shown), indicating that the induction of MUC5AC production by the recombinant S100 proteins was not due to endotoxin contamination. We also tested the specificity of the three S100 proteins using blocking antibodies. Treatment with the blocking antibodies resulted in significant decreases in both MUC5AC mRNA and protein expression (Fig. 2c, d). Taken together, these data corroborated the bona fide ability of the three S100 proteins to induce MUC5AC production.

Figure 1.

Figure 1

Open in a new tab

Induction of MUC5AC mRNA and protein in airway epithelial cells by S100 proteins. (a) NCI-H292 cells were treated with the indicated concentrations of S100A8, S100A9 and S100A12 (0–500 ng/ml), and epidermal gorwth factor (EGF; 30 ng/ml) for 8 hr, and analysed for MUC5AC mRNA expression by real-time PCR. GAPDH was used to normalize levels of MUC5AC transcripts. Data are shown as the mean ± SEM of four independent experiments performed in triplicate (*P < 0·05 and **P < 0·01 compared with unstimulated cells). (b) NCI-H292 cells were treated with S100A8, S100A9 and S100A12 (each 200 ng/ml) for 0, 4, 8, 12 and 24 hr. Data are shown as the mean ± SEM of four independent experiments performed in triplicate (**P < 0·01 compared with unstimulated cells at 0 hr). (c) NCI-H292 cells were treated with the three S100 proteins (each 200 ng/ml) for 24 hr, and were subjected to immunocytochemistry (upper row) and immunofluorescent staining (lower row) with an anti-MUC5AC antibody as described in the Materials and methods. (d) NCI-H292 cells were treated with various concentrations of S100A8, S100A9 and S100A12 (0–200 ng/ml), and EGF (30 ng/ml) for 24 hr, and analysed for MUC5AC protein expression by immunocytochemistry. MUC5AC-positive cells were enumerated and expressed as the percentage of total cells. Data are shown as the mean ± SEM of three independent experiments (*P < 0·05 and **P < 0·01 compared with unstimulated cells). (e) NCI-H292 cells were transfected with a MUC5AC promoter reporter. After 24 hr, the cells were treated with the S100 proteins (200 ng/ml) for 12 hr, lysed and assayed for dual luciferase activities. Data are shown as the means ± SEM of six independent experiments performed in triplicate (**P < 0·01 compared with unstimulated cells). (f) Normal human bronchial epithelial (NHBE) cells were treated with the three S100 proteins (0–500 ng/ml) and EGF (30 ng/ml) for 8 hr and analysed for MUC5AC mRNA by real-time PCR. Data are shown as the mean ± SEM of 6–12 independent experiments performed in triplicate (*P < 0·05 and **P < 0·01 compared with unstimulated cells).

Figure 2.

Figure 2

Open in a new tab

Specificity of S100 protein activity to induce MUC5AC production. (a, b) NCI-H292 cells were treated with the three S100 proteins S100A8, S100A9 and S100A12 (200 ng/ml) in the presence or absence of polymyxin B (10 μg/ml) for 8 and 24 hr for MUC5AC mRNA and protein expression, respectively. Data are representative of three independent experiments performed and are shown as the mean ± SEM. (c, d) Neutralizing antibodies (0·5 μg/ml) of the three S100 proteins or their control antibodies (0·5 μg/ml) were allowed to bind the three S100 proteins for 30 min. NCI-H292 cells were then treated with the mixtures and MUC5AC production was determined. Data are shown as the mean ± SEM of three independent experiments performed (*P < 0·05 and **P < 0·01, neutralizing antibodies versus control antibodies). For MUC5AC mRNA, real-time PCR was carried out in triplicate (a, c).

S100A8- and S100A9-induced MUC5AC expression is TLR4-dependent, whereas S100A12-induced MUC5AC expression is both TLR4- and RAGE-dependent

We next examined whether MUC5AC expression by the three S100 proteins occurred through two well-known multi-ligand receptors TLR420 and RAGE.21 TLR4 and RAGE mRNAs were constitutively and abundantly expressed in both NCI-H292 cells and NHBE cells, and remained unaltered in response to treatment with the three S100 proteins (see Supporting information, Fig. S3). Treatment with TAK-242, a TLR4 inhibitor, almost completely abolished MUC5AC mRNA and protein expression by all three S100 proteins. In addition, neutralization of RAGE by its blocking antibody led to a significant inhibition of S100A12-mediated MUC5AC expression, but did not affect S100A8- or S100A9-mediated production (Fig. 3). These data conclusively indicated that S100A8-mediated and S100A9-mediated MUC5AC production in airway epithelial cells occurred through TLR4 but not RAGE, while the effects of S100A12 required both TLR4 and RAGE.

Figure 3.

Figure 3

Open in a new tab

MUC5AC expression through Toll-like receptor 4 (TLR4) for S100A8, S100A9 and S100A12 and through receptor for advanced glycation end-products (RAGE) for S100A12. NCI-H292 cells were pre-treated with the TLR4 inhibitor TAK-242 (1 μg/ml) and anti-RAGE antibody (5 μg/ml) for 30 min followed by treatment with the three S100 proteins for 8 and 24 hr to determine MUC5AC mRNA and protein expression, respectively. (a) The expression of MUC5AC mRNA was analysed by RT-PCR and real-time PCR; the latter data are shown as the mean ± SEM of four independent experiments performed in triplicate (*P < 0·05 and **P < 0·01 compared with unstimulated cells). (b) The expression of MUC5AC protein was analysed by immunocytochemistry. Data are shown as the mean ± SEM of three independent experiments (*P < 0·05 and **P < 0·01 compared with unstimulated cells).

S100A8, S100A9 and S100A12 induce MUC5AC expression through ERK and NF-κB pathways

Using various pharmacological inhibitors, we next examined signalling pathways that might be responsible for the induction of MUC5AC expression by S100 proteins. S100A8-induced MUC5AC mRNA and protein expression were nearly completely inhibited by EGFR inhibitor AG1478, NF-κB inhibitor BAY11, ERK inhibitor U0126, p38 inhibitor SB203580, and JNK inhibitor SP600125 (Fig. 4a, b). Similarly, these drugs also inhibited S100A9-induced or S100A12-induced MUC5AC mRNA expression (Fig. 4a). Treatment of NHBE cells with any of the four different inhibitors AG1478, BAY11 or U0126 almost completely inhibited MUC5AC mRNA expression (Fig. 4c). These results suggested that S100 protein-induced MUC5AC expression was mediated through signalling pathways, including mitogen-activated protein kinases, NF-κB and EGFR pathways.

Figure 4.

Figure 4

Open in a new tab

Involvement of epidermal growth factor (EGFR), nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signalling in S100A8-induced MUC5AC expression. NCI-H292 and normal human bronchial epithelial (NHBE) cells were pre-treated with AG1478, BAY11, U0126, SB203580 and SP600125 for 30 min and then treated with these S100 proteins (200 ng/ml each) for 8 and 24 hr for MUC5AC mRNA and protein expression, respectively. The inhibitors were used at a concentration of 10 μm, except for SP600125, which was used at 20 μm. (a) The expression of MUC5AC mRNA was analysed by RT-PCR and real-time PCR. The latter data are shown as the means ± SEM of three independent experiments performed in triplicate (**P < 0·01, compared with S100 protein-treated cells). (b) MUC5AC-positive cells were enumerated. Data are shown as the mean ± SEM of three independent experiments (**P < 0·01 compared with S100A8-treated cells). (c) The expression of MUC5AC mRNA in NHBE cells was analysed by real-time PCR. Data are shown as means ± SEM of six independent experiments performed in triplicate (*P < 0·05 and **P < 0·01, compared with S100A8-treated cells).

S100A8, S100A9 and S100A12 activate the ERK pathway in airway epithelial cells

Numerous studies have demonstrated that mitogen-activated protein kinases and NF-κB are involved in regulating MUC5AC expression.11 Hence, we examined whether the activity of S100 proteins to induce MUC5AC expression occurred through engagement of the aforementioned pathways at the biochemical level. A basal level of phosphorylated ERK1/2 was detected in unstimulated NCI-H292 cells. Following stimulation with S100A8, ERK phosphorylation was markedly increased, with peak levels of phosphorylation appearing at 6–8 hr. Phosphorylated JNK was maintained at a relatively high level in unstimulated cells, and was slightly augmented following stimulation with S100A8 for 6–8 hr. In contrast, levels of phosphorylated p38 remained unchanged before and after stimulation (Fig. 5a). At this moment, it is not clear why there is a lack of consistency between the effect of the p38 inhibitor on S100A8-mediated MUC5AC expression and the level of p38 phosphorylation in cells stimulated with S100A8. In addition to S100A8, S100A9 and S100A12 markedly stimulated ERK phosphorylation (Fig. 5b). Furthermore, the three S100 proteins strongly stimulated ERK phosphorylation in NHBE cells (Fig. 5c). Phosphorylation of EGFR, which is upstream of ERK,14 was also observed in NCI-H292 and NHBE cells in response to the three S100 proteins (Fig. 5b, c). When NCI-H292 cells were treated with S100A8 in the presence of different inhibitors for EGFR, NF-κB, or ERK, ERK phosphorylation was inhibited (Fig. 5d) in each case, suggesting that signals for MUC5AC expression converged on the ERK pathway. Taken together, these results suggested that all three S100A proteins activate the ERK pathway in airway epithelial cells.

Figure 5.

Figure 5

Open in a new tab

Activation of mitogen-activated protein kinases (MAPKs) and epidermal growth factor receptor (EGFR) by S100 proteins. (a) NCI-H292 cells were stimulated with S100A8 (200 ng/ml) for the indicated time intervals and 20 μg of protein of cell lysates was analysed for phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK). To control for equal loading, the same blots were re-probed with the indicated antibodies. (b, c) NCI-H292 cells and normal human bronchial epithelial (NHBE) cells were treated with S100A8, S100A9 and S100A12 (each 200 ng/ml) for 8 hr and probed for phosphorylation of ERK and EGFR. (d) NCI-H292 cells were pre-treated with AG1478, BAY11, and U0126 for 30 min, and then treated with S100A8 for 8 hr. The blot was probed with anti-phospho-ERK1/2 antibody. Blots are representatives of two to three independent experiments.

S100A8, S100A9 and S100A12 activate the NF-κB pathway in airway epithelial cells

Nuclear factor-κB serves a key molecule that mediates TLR4 and RAGE pathways.21,22 As MUC5AC expression by the S100 proteins was almost completely abrogated by blockage of TLR4 and RAGE (Fig. 3), and further by the NF-κB inhibitor BAY11 (Fig. 4), we next examined NF-κB activation by the S100 proteins in NCI-H292 and NHBE cells. NCI-H292 cells were transfected with an NF-κB-hrGFP reporter plasmid and then stimulated with S100A8, S100A9 and S100A12. All three S100 proteins robustly activated an NF-κB-hrGFP reporter to a level similar to that produced by lipopolysaccharide, which was used as a positive control (Fig. 6a). A similar result was obtained with the NF-κB-Luc reporter plasmid (Fig. 7c). It was noteworthy that the three S100 proteins activated the NF-κB-Luc reporter to a level similar to that with lipopolysaccharide (see Fig. 7c). Consistent with a previous report,12 EGF failed to activate the NF-κB-hrGFP reporter in NCI-H292 cells (Fig. 6a) and so served to validate the assay. Biochemical analysis demonstrated that the nuclear translocation of NF-κB was increased up to 8 hr following treatment with S100A8. With similar kinetic behaviour, levels of IκB also decreased gradually (Fig. 6b). Likewise, both S100A9 and S10012 augmented nuclear translocation of NF-κB in NHBE cells as well as in NCI-H293 cells (Fig. 6c) similar to S100A8. Collectively, these results demonstrated activation of the NF-κB pathway by the three S100 proteins.

Figure 6.

Figure 6

Open in a new tab

Activation of nuclear factor-κB (NF-κB) by S100 proteins. (a) NCI-H292 cells were transfected with 2 μg of NF-κB-hrGFP reporter plasmid. After 24 hr, the cells were treated with the three S100A proteins (200 ng/ml), lipopolysaccharide (LPS; 100 ng/ml), and epidermal growth factor (EGF; 30 ng/ml) and incubated for an additional 8 hr. Fluorescent microscopic data are representative of three independent experiments. (b) NCI-H292 cells were treated with S100A8 for the indicated times (0–12 hr). Cytosol and nuclear fractions were isolated for determination of IκB and NF-κB (p65) expression, respectively. extracellular signal-regulated kinase 2 (ERK2) and Histone3 were used as loading controls for cytosolic and nuclear proteins, respectively. The blots shown are representative of three independent experiments. (c) NCI-H292 cells and normal human bronchial epithelial (NHBE) cells were treated with three S100A proteins for 8 hr and analysed for nuclear localization of NF-κB (p65). The blots shown are representatives of three or more independent experiments.

Figure 7.

Figure 7

Open in a new tab

Effects of Toll-like receptor 4 (TLR4) and receptor for advanced glycation end-products (RAGE) blockage on S100 protein-mediated activation of extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB). (a, b) NCI-H292 cells were pre-treated with the TLR4 inhibitor TAK-242 (1 μg/ml) or anti-RAGE antibody (10 μg/ml) for 30 min and then treated with the three S100 proteins for 8 hr. Total cell lysates (20 μg) and nuclear extracts (10 μg) were analysed for phosphorylation of ERK1/2 and nuclear translocation of NF-κB, respectively. ERK2 and Histone3 were used as loading controls for cytosolic and nuclear proteins, respectively. Relative intensities were analysed by densitometry and expressed as mean ± SEM of three independent experiments (*P < 0·05 and **P < 0·01, compared with the S100 protein-treated cells). Representative blots are shown. (c) NCI-H292 cells were transfected with the NF-κB Luc reporter plasmid and treated with S100 proteins in the presence of TAK-242 or anti-RAGE antibody for 8 hr. Data are the mean ± SEM of two independent experiments performed in triplicate (**P < 0·01 compared with S100 protein-treated cells).

S100A8, S100A9 and S100A12 activate ERK and NF-κB via TLR4 and RAGE

To assess the relative contributions of TLR4 and RAGE towards activation of the ERK and NF-κB pathways induced by the three S100 proteins, NCI-H292 cells were pre-treated with TAK-242 or an anti-RAGE antibody before addition of the three S100 proteins, after which phosphorylation of ERK and nuclear translocation of NF-κB were analysed. Blocking TLR4 activity inhibited ERK phosphorylation elicited by all three S100 proteins (Fig. 7a, lane 2 versus lane 3 for S100A8, lane 5 versus lane 6 for S100A9, and lane 8 versus lane 9 for S100A12). In exact concordance, when the cells were stimulated with the three S100 proteins in the presence of the TLR4 inhibitor, nuclear translocation of NF-κB was severely inhibited (Fig. 7b). When RAGE was neutralized in cells treated with S100A12, ERK phosphorylation was inhibited (lane 8 versus lane 10 in Fig. 7a), whereas nuclear translocation of NF-κB was affected only minimally (lane 8 versus lane 10 in Fig. 7b). S100A12-induced ERK phosphorylation was inhibited by anti-RAGE in a dose-dependent manner (see Supporting information, Fig. S4). In addition, neutralization of RAGE had no effect on either the ERK or NF-κB pathways that were activated by S100A8 and S100A9 (Fig. 7a, b, lanes 2 versus lanes 4 and lanes 5 versus lanes 7). To further verify the differential effect of these three S100 proteins on NF-κB activation through TLR4 and RAGE, NCI-H292 cells were transfected with an NF-κB-Luc reporter, treated with the S100 proteins in the presence of TAK-242 or anti-RAGE antibody for 8 hr, and assayed for reporter activity. An almost identical pattern was observed with respect to NF-κB activity as demonstrated by nuclear translocation of NF-κB: a significant reduction in S100A8-mediated and S100A9-mediated NF-κB activity by TAK-242 but not by an anti-RAGE antibody; a significant effect by TAK-242, and a marginal effect by an anti-RAGE antibody on S100A12-mediated NF-κB activity (Fig. 7c). Collectively, these data indicated that all three S100 proteins strongly activated both ERK and NF-κB pathways through TLR4, whereas S10012 activated the ERK pathway rather than the NF-κB pathway through RAGE in lung epithelial cells.

Discussion

Neutrophilic airway inflammation is strongly associated with severe, steroid-resistant asthma, COPD and exacerbation of these diseases.33 S100A8, S100A9 and S100A12 belong to the DAMP family members of danger signals that initiate and amplify local inflammation and innate immune responses.18,20,23 These S100 proteins are present in the greatest abundance in neutrophils and have been implicated in the pathogenesis of a number of inflammatory diseases. Furthermore, these S100 proteins have been proposed as key contributors to the pathological development of obstructive pulmonary diseases.34 Accordingly, we hypothesized that they could induce expression of MUC5AC, a major mucin in conducting airways, whose overproduction is one of the most critical parameters in the pathophysiological processes of obstructive airway disease. Our results demonstrated that the three S100 proteins stimulated airway epithelial cells to induce expression of MUC5AC mRNA and protein and activate a MUC5AC promoter reporter. Blockage of two prominent receptors, TLR4 and RAGE, abrogated S100 protein-mediated expression of MUC5AC in a manner that depends on the characteristic interactions between these S100 proteins and the two receptors. In addition, ERK and NF-κB pathways, which represent the two major signalling pathways that are activated following engagement of the TLR4 and RAGE receptors by the three S100 proteins, were essential for S100 protein-mediated MUC5AC expression. Collectively, our results for the first time identify a new role of S100A8, S100A9 and S100A12 for MUC5AC production by acting on airway epithelial cells, and provide a molecular basis for the role of these three S100 proteins in the pathogenesis of neutrophil-dominant obstructive airway diseases.

The three S100 proteins S100A8, S100A9 and S100A12 are released into the extracellular compartment due to cell damage, infection and inflammation, or are secreted by activated cells. S100A8, S100A9 and S100A12 mediate inflammatory responses by acting on a variety of immune and non-immune cells, including monocytes, neutrophils, endothelial cells, keratinocytes, epithelial cells and tumour cells. The released S100 proteins induce production of pro-inflammatory cytokines,20,21,35–38 neutrophil degranulation and chemotaxis,38,39 leucocyte adhesion and endothelial transmigration,40 increased effects of lipopolysaccharide on phagocytes,20,23 and proliferation of several cell types.37,41 The pro-inflammatory response to S100 proteins occurs through TLR4 and RAGE. TLR4 is expressed in bronchial epithelial cells and mediates lipopolysaccharide-induced inflammatory responses,42,43 whereas RAGE is expressed at a low level in bronchial epithelial cells of normal human lungs but is up-regulated under pathological conditions.44 Interestingly, mice lacking RAGE exhibit reduced lung inflammation.45 We found a high level of constitutively expressed TLR4 and RAGE mRNA in the mucus-secreting cells NCI-H292 and NHBE cells (see Supporting information, Fig. S4). These results, along with the observation that the blockage of the two receptors resulted in ablated or diminished MUC5AC production (Fig. 3), indicated that the three S100 proteins S100A8, S100A9 and S100A12 were specifically capable of activating airway epithelial cells to stimulate production of MUC5AC. In contrast, HMGB1, the prototype DAMP molecule, was unable to induce MUC5AC expression in airway epithelial cells (ref.46 and our unpublished observations), despite sharing many aspects with the S100 proteins, including receptor usage, signalling pathways and pro-inflammatory functions. Hence, the ability to induce MUC5AC production appears to be unique to S100 proteins, which has important implications in the context of lung inflammation associated with airway obstruction.

A number of studies have demonstrated specific interactions between the three S100 proteins and their receptors TLR4 and RAGE in a variety of cell types. It is widely accepted that S100A8, S100A9 and their heterodimer are specific for TLR4,20,47,48 whereas S100A12 is specific for RAGE.21,49 Nonetheless, there have been several reports suggesting that S100A8/S100A9 and S100A12 can bind to and cross-activate RAGE and TLR4, respectively. For example, S100A9 activates ERK1/2 and NF-κB pathways in human lung fibroblast cells through RAGE ligation,37 and the S100A8/A9 heterodimer promotes the growth of tumour cells in a RAGE-dependent manner.24 Both S100A8 and S100A9 trigger translocation of RAGE in human prostate cancer cells.25 S100A9, but neither S100A8 nor the heterodimer, binds RAGE as well as TLR4 in vitro.50 Reciprocally, a recent finding showed an interaction between S100A12 and TLR4.51 In that particular study, blocking S100A12 binding to TLR4, but not RAGE, on monocytes or TLR4-expressing cell lines abrogated inflammatory signalling. The results of our study showed that cross-activation was partly responsible for MUC5AC production induced by the three S100 proteins in airway epithelial cells. Specifically, we observed that S100A8 and S100A9 could not activate RAGE, while S100A12 activates TLR4 to produce MUC5AC in airway epithelial cells (Fig. 3). In addition, with respect to activation of the S100A12-RAGE axis, it was noteworthy that blocking of S100A12 binding to RAGE preferentially affected ERK rather than NF-κB activation (Fig. 7a, b). The inability of S100A12 to mediate NF-κB nuclear translocation through RAGE was further supported by the fact that activity of the NF-κB-Luc reporter was only marginally affected by an anti-RAGE antibody in cells treated with S100A12 (Fig. 7c). The results from the previous studies and this study suggest the possibility of more flexible interactions between the three S100 proteins and the RAGE and TLR4 receptors compared with the established relationships between the S100A8/S100A9-TLR4 and S100A12-RAGE axes. It is therefore presumed that the interactions of the S100 proteins S100A8, S100A9, and S100A12 with the RAGE and TLR4 receptors may depend greatly on cell type and/or inflammatory responses.

Neutrophil elastase is an abundant extracellular product of neutrophils along with the S100 proteins and is associated with many obstructive airway diseases largely by facilitating production of MUC5AC.7,8 The NE activates airway epithelial cells to induce MUC5AC production in a manner that is different from the mode of action of S100 proteins. Specifically, NE activates TNF-α converting enzyme via reactive oxygen species generation, leading to production of transforming growth factor-α and activation of EGFR resulting in MUC5AC production.52 Given the effects of the S100 proteins S100A8, S100A9 and S100A12 observed in our study, they must now be recognized as new members of the set of neutrophil products able to induce MUC5AC production. Some of the three S100 genes or proteins are over-expressed or abundantly present in sites of inflammation and body fluid in airway diseases.27,28,53 Indeed, S100 proteins trigger MUC5AC production at concentrations (a few dozen nM) similar to those at which they are present in airway secretions of individuals with obstructive airway diseases.28,53 In addition, the concentrations of the S100 proteins were similar to that of NE needed to provoke MUC5AC production.52 Hence, our data suggest that, much like NE, the three S100 proteins S100A8, S100A9 and S100A12 play a crucial role in the pathogenesis of the obstructive pulmonary diseases by causing MUC5AC production. Determining the relative abundance of S100 proteins compared to NE in neutrophilic inflammations will help to evaluate the relative contributions and potency of these two classes of neutrophil products to induce production of MUC5AC.

Our data showed that the S100 protein-mediated MUC5AC expression was inhibited by an EGFR inhibitor (Fig. 4). In line, the S100 proteins activated EGFR in both NCI-H292 and NHBE cells (Fig. 5). In this sense, NE appears to act in analogous manners. The NE activates not only TLR4 but also EGFR, which is activated via generation of reactive oxygen species and transforming growth factor-α.52 Furthermore, EGFR co-localizes with TLR4 in airway epithelial cells.54 It raises the possibility that these S100 proteins-activated EGFR and TLR4 might engage in cross-talk to activate intracellular signals leading to a change in MUC5AC expression.

In summary, we identified an activity of the S100 proteins S100A8, S100A9 and S100A12 to induce MUC5AC production in airway epithelial cells through ligations with the receptors TLR4 and RAGE. A strong association has been established between airway neutrophilia and obstructive airway diseases such as severe asthma, COPD and cystic fibrosis. Hence, it is important to identify mediators released by neutrophils that are unresponsive to steroid therapy. Given that these three S100 proteins are among the most abundant proteins expressed by neutrophils, the results of this study reveal a link between chronic neutrophilic inflammation and increased mucin production in airway diseases. These results may also help to further our understanding of the pathogenesis of neutrophil-dominant airway inflammation. Accordingly, these S100 proteins described here constitute new therapeutic targets for obstructive airway diseases associated with neutrophilic inflammation.

Acknowledgments

This research was supported by the Basic Science Research Programme through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2041988 to JHK). We thank Dr Philippe Tessier at Laval University, Canada for providing blocking anti-S100A8 and anti-S100A9 antibodies.

Disclosures

The authors declare no financial and commercial conflicts of interest.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1. Induction of MUC5AC secretion by S100 protein in normal human bronchial epithelial cells.

imm0144-0079-sd1.pdf (10.3KB, pdf)

Figure S2. Induction of MUC5AC protein by S100A8 in normal human bronchial epithelial cells.

imm0144-0079-sd2.pdf (20.4KB, pdf)

Figure S3. The expression of TLR4 and RAGE mRNAs in airway epithelial cells.

imm0144-0079-sd3.pdf (31.8KB, pdf)

Figure S4. Effect of anti-receptor for advanced glycation end-products (RAGE) antibody on S100A12-mediated activation of extracellular signal-regulated kinase.

imm0144-0079-sd4.pdf (36.2KB, pdf)
imm0144-0079-sd5.doc (32KB, doc)

References

  • 1.Jatakanon A, Uasuf C, Maziak W, Lim S, Chung KF, Barnes PJ. Neutrophilic inflammation in severe persistent asthma. Am J Respir Crit Care Med. 1999;160:1532–9. doi: 10.1164/ajrccm.160.5.9806170. [DOI] [PubMed] [Google Scholar]
  • 2.Wenzel SE, Schwartz LB, Langmack EL, Halliday JL, Trudeau JB, Gibbs RL, Chu HW. Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics. Am J Respir Crit Care Med. 1999;160:1001–8. doi: 10.1164/ajrccm.160.3.9812110. [DOI] [PubMed] [Google Scholar]
  • 3.Hoenderdos K, Condliffe A. The neutrophil in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol. 2013;48:531–9. doi: 10.1165/rcmb.2012-0492TR. [DOI] [PubMed] [Google Scholar]
  • 4.Gibson RL, Burns JL, Ramsey BW. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am J Respir Crit Care Med. 2003;168:918–51. doi: 10.1164/rccm.200304-505SO. [DOI] [PubMed] [Google Scholar]
  • 5.Armstrong J, Sargent C, Singh D. Glucocorticoid sensitivity of lipopolysaccharide-stimulated chronic obstructive pulmonary disease alveolar macrophages. Clin Exp Immunol. 2009;158:74–83. doi: 10.1111/j.1365-2249.2009.03986.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Amulic B, Cazalet C, Hayes GL, Metzler KD, Zychlinsky A. Neutrophil function: from mechanisms to disease. Annu Rev Immunol. 2012;30:459–89. doi: 10.1146/annurev-immunol-020711-074942. [DOI] [PubMed] [Google Scholar]
  • 7.Nadel JA, Takeyama K, Agustí C. Role of neutrophil elastase in hypersecretion in asthma. Eur Respir J. 1999;13:190–6. doi: 10.1034/j.1399-3003.1999.13a35.x. [DOI] [PubMed] [Google Scholar]
  • 8.Voynow JA, Young LR, Wang Y, Horger T, Rose MC, Fischer BM. Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells. Am J Physiol. 1999;276:L835–43. doi: 10.1152/ajplung.1999.276.5.L835. [DOI] [PubMed] [Google Scholar]
  • 9.Zuhdi Alimam M, Piazza FM, Selby DM, Letwin N, Huang L, Rose MC. Muc-5/5ac mucin messenger RNA and protein expression is a marker of goblet cell metaplasia in murine airways. Am J Respir Cell Mol Biol. 2000;22:253–60. doi: 10.1165/ajrcmb.22.3.3768. [DOI] [PubMed] [Google Scholar]
  • 10.Ordoñez CL, Khashayar R, Wong HH, et al. Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression. Am J Respir Crit Care Med. 2001;163:517–23. doi: 10.1164/ajrccm.163.2.2004039. [DOI] [PubMed] [Google Scholar]
  • 11.Thai P, Loukoianov A, Wachi S, Wu R. Regulation of airway mucin gene expression. Annu Rev Physiol. 2008;70:405–29. doi: 10.1146/annurev.physiol.70.113006.100441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Lora JM, Zhang DM, Liao SM, et al. Tumor necrosis factor-α triggers mucus production in airway epithelium through an IκB kinase β-dependent mechanism. J Biol Chem. 2005;280:36510–7. doi: 10.1074/jbc.M507977200. [DOI] [PubMed] [Google Scholar]
  • 13.Chen R, Lim JH, Jono H, Gu XX, Kim YS, Basbaum CB, Murphy TF, Li JD. Nontypeable Haemophilus influenzae lipoprotein P6 induces MUC5AC mucin transcription via TLR2-TAK1-dependent p38 MAPK-AP1 and IKKβ-IκBα-NF-κB signaling pathways. Biochem Biophys Res Commun. 2004;324:1087–94. doi: 10.1016/j.bbrc.2004.09.157. [DOI] [PubMed] [Google Scholar]
  • 14.Perrais M, Pigny P, Copin MC, Aubert JP, Van Seuningen I. Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/Ras/Raf/extracellular signal-regulated kinase cascade and Sp1. J Biol Chem. 2002;277:32258–67. doi: 10.1074/jbc.M204862200. [DOI] [PubMed] [Google Scholar]
  • 15.Song KS, Lee WJ, Chung KC, Koo JS, Yang EJ, Choi JY, Yoon JH. Interleukin-1 β and tumor necrosis factor-α induce MUC5AC overexpression through a mechanism involving ERK/p38 mitogen-activated protein kinases-MSK1-CREB activation in human airway epithelial cells. J Biol Chem. 2003;278:23243–50. doi: 10.1074/jbc.M300096200. [DOI] [PubMed] [Google Scholar]
  • 16.Hewson CA, Edbrooke MR, Johnston SL. PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-α, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms. J Mol Biol. 2004;344:683–95. doi: 10.1016/j.jmb.2004.09.059. [DOI] [PubMed] [Google Scholar]
  • 17.Li D, Gallup M, Fan N, Szymkowski DE, Basbaum CB. Cloning of the amino-terminal and 5’-flanking region of the human MUC5AC mucin gene and transcriptional up-regulation by bacterial exoproducts. J Biol Chem. 1998;273:6812–20. doi: 10.1074/jbc.273.12.6812. [DOI] [PubMed] [Google Scholar]
  • 18.Foell D, Wittkowski H, Vogl T, Roth J. S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J Leukoc Biol. 2007;81:28–37. doi: 10.1189/jlb.0306170. [DOI] [PubMed] [Google Scholar]
  • 19.Edgeworth J, Gorman M, Bennett R, Freemont P, Hogg N. Identification of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells. J Biol Chem. 1991;266:7706–13. [PubMed] [Google Scholar]
  • 20.Vogl T, Tenbrock K, Ludwig S, et al. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock. Nat Med. 2007;13:1042–9. doi: 10.1038/nm1638. [DOI] [PubMed] [Google Scholar]
  • 21.Hofmann MA, Drury S, Fu C, et al. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell. 1999;97:889–901. doi: 10.1016/s0092-8674(00)80801-6. [DOI] [PubMed] [Google Scholar]
  • 22.Huttunen HJ, Kuja-Panula J, Sorci G, Agneletti AL, Donato R, Rauvala H. Coregulation of neurite outgrowth and cell survival by amphoterin and S100 proteins through receptor for advanced glycation end products (RAGE) activation. J Biol Chem. 2000;275:40096–105. doi: 10.1074/jbc.M006993200. [DOI] [PubMed] [Google Scholar]
  • 23.Ehrchen JM, Sunderkötter C, Foell D, Vogl T, Roth J. The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer. J Leukoc Biol. 2009;86:557–66. doi: 10.1189/jlb.1008647. [DOI] [PubMed] [Google Scholar]
  • 24.Ghavami S, Rashedi I, Dattilo BM, et al. S100A8/A9 at low concentration promotes tumor cell growth via RAGE ligation and MAP kinase-dependent pathway. J Leukoc Biol. 2008;83:1484–92. doi: 10.1189/jlb.0607397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Hermani A, De Servi B, Medunjanin S, Tessier PA, Mayer D. S100A8 and S100A9 activate MAP kinase and NF-κB signaling pathways and trigger translocation of RAGE in human prostate cancer cells. Exp Cell Res. 2006;312:184–97. doi: 10.1016/j.yexcr.2005.10.013. [DOI] [PubMed] [Google Scholar]
  • 26.Yang Z, Yan WX, Cai H, et al. S100A12 provokes mast cell activation: a potential amplification pathway in asthma and innate immunity. J Allergy Clin Immunol. 2007;119:106–14. doi: 10.1016/j.jaci.2006.08.021. [DOI] [PubMed] [Google Scholar]
  • 27.Aoki T, Matsumoto Y, Hirata K, et al. Expression profiling of genes related to asthma exacerbations. Clin Exp Allergy. 2009;39:213–21. doi: 10.1111/j.1365-2222.2008.03186.x. [DOI] [PubMed] [Google Scholar]
  • 28.Lee TH, Jang AS, Park JS, et al. Elevation of S100 calcium binding protein A9 in sputum of neutrophilic inflammation in severe uncontrolled asthma. Ann Allergy Asthma Immunol. 2013;111:268–75. doi: 10.1016/j.anai.2013.06.028. [DOI] [PubMed] [Google Scholar]
  • 29.Vandal K, Rouleau P, Boivin A, Ryckman C, Talbot M, Tessier PA. Blockade of S100A8 and S100A9 suppresses neutrophil migration in response to lipopolysaccharide. J Immunol. 2003;171:2602–9. doi: 10.4049/jimmunol.171.5.2602. [DOI] [PubMed] [Google Scholar]
  • 30.Kang JH, Lee EH, Park SW, Chung IY. MUC5AC expression through bidirectional communication of Notch and epidermal growth factor receptor pathways. J Immunol. 2011;187:222–9. doi: 10.4049/jimmunol.1003606. [DOI] [PubMed] [Google Scholar]
  • 31.Song JS, Cho KS, Yoon HK, Moon HS, Park SH. Neutrophil elastase causes MUC5AC mucin synthesis via EGF receptor, ERK and NF-κB pathways in A549 cells. Korean J Intern Med. 2005;20:275–83. doi: 10.3904/kjim.2005.20.4.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Kawai T, Akira S. TLR signaling. Cell Death Differ. 2006;13:816–25. doi: 10.1038/sj.cdd.4401850. [DOI] [PubMed] [Google Scholar]
  • 33.Cowburn AS, Condliffe AM, Farahi N, Summers C, Chilvers ER. Advances in neutrophil biology: clinical implications. Chest. 2008;134:606–12. doi: 10.1378/chest.08-0422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Halayko AJ, Ghavami S. S100A8/A9: a mediator of severe asthma pathogenesis and morbidity? Can J Physiol Pharmacol. 2009;87:743–55. doi: 10.1139/Y09-054. [DOI] [PubMed] [Google Scholar]
  • 35.Sunahori K, Yamamura M, Yamana J, et al. The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor κB and p38 mitogen-activated protein kinase in rheumatoid arthritis. Arthritis Res Ther. 2006;8:R69. doi: 10.1186/ar1939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Riva M, Källberg E, Björk P, Hancz D, Vogl T, Roth J, Ivars F, Leanderson T. Induction of nuclear factor-κB responses by the S100A9 protein is Toll-like receptor-4-dependent. Immunology. 2012;137:172–82. doi: 10.1111/j.1365-2567.2012.03619.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Xu X, Chen H, Zhu X, et al. S100A9 promotes human lung fibroblast cells activation through receptor for advanced glycation end-product-mediated extracellular-regulated kinase 1/2, mitogen-activated protein-kinase and nuclear factor-κB-dependent pathways. Clin Exp Immunol. 2013;173:523–35. doi: 10.1111/cei.12139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Simard JC, Girard D, Tessier PA. Induction of neutrophil degranulation by S100A9 via a MAPK-dependent mechanism. J Leukoc Biol. 2010;87:905–14. doi: 10.1189/jlb.1009676. [DOI] [PubMed] [Google Scholar]
  • 39.Ryckman C, Vandal K, Rouleau P, Talbot M, Tessier PA. Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion. J Immunol. 2003;170:3233–42. doi: 10.4049/jimmunol.170.6.3233. [DOI] [PubMed] [Google Scholar]
  • 40.Viemann D, Strey A, Janning A, et al. Myeloid-related proteins 8 and 14 induce a specific inflammatory response in human microvascular endothelial cells. Blood. 2005;105:2955–62. doi: 10.1182/blood-2004-07-2520. [DOI] [PubMed] [Google Scholar]
  • 41.Nukui T, Ehama R, Sakaguchi M, Sonegawa H, Katagiri C, Hibino T, Huh NH. S100A8/A9, a key mediator for positive feedback growth stimulation of normal human keratinocytes. J Cell Biochem. 2008;104:453–64. doi: 10.1002/jcb.21639. [DOI] [PubMed] [Google Scholar]
  • 42.Guillot L, Medjane S, Le-Barillec K, Balloy V, Danel C, Chignard M, Si-Tahar M. Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways: evidence for an intracellular compartmentalization of TLR4. J Biol Chem. 2004;279:2712–8. doi: 10.1074/jbc.M305790200. [DOI] [PubMed] [Google Scholar]
  • 43.MacRedmond R, Greene C, Taggart CC, McElvaney N, O'Neill S. Respiratory epithelial cells require Toll-like receptor 4 for induction of human β-defensin 2 by lipopolysaccharide. Respir Res. 2005;6:116. doi: 10.1186/1465-9921-6-116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Morbini P, Villa C, Campo I, Zorzetto M, Inghilleri S, Luisetti M. The receptor for advanced glycation end products and its ligands: a new inflammatory pathway in lung disease? Mod Pathol. 2006;19:1437–45. doi: 10.1038/modpathol.3800661. [DOI] [PubMed] [Google Scholar]
  • 45.van Zoelen MA, Yang H, Florquin S, et al. Role of toll-like receptors 2 and 4, and the receptor for advanced glycation end products in high-mobility group box 1-induced inflammation in vivo. Shock. 2009;31:280–4. doi: 10.1097/SHK.0b013e318186262d. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Kim DE, Min KJ, Kim JS, Kwon TK. High-mobility group box-1 protein induces mucin 8 expression through the activation of the JNK and PI3K/Akt signal pathways in human airway epithelial cells. Biochem Biophys Res Commun. 2012;421:436–41. doi: 10.1016/j.bbrc.2012.03.131. [DOI] [PubMed] [Google Scholar]
  • 47.Petersen B, Wolf M, Austermann J, et al. The alarmin Mrp8/14 as regulator of the adaptive immune response during allergic contact dermatitis. EMBO J. 2013;32:100–11. doi: 10.1038/emboj.2012.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Loser K, Vogl T, Voskort M, et al. The Toll-like receptor 4 ligands Mrp8 and Mrp14 are crucial in the development of autoreactive CD8+ T cells. Nat Med. 2010;16:713–7. doi: 10.1038/nm.2150. [DOI] [PubMed] [Google Scholar]
  • 49.Srikrishna G, Nayak J, Weigle B, et al. Carboxylated N-glycans on RAGE promote S100A12 binding and signaling. J Cell Biochem. 2010;110:645–59. doi: 10.1002/jcb.22575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Björk P, Björk A, Vogl T, et al. Identification of human S100A9 as a novel target for treatment of autoimmune disease via binding to quinoline-3-carboxamides. PLoS Biol. 2009;7:e97. doi: 10.1371/journal.pbio.1000097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Foell D, Wittkowski H, Kessel C, et al. Proinflammatory S100A12 can activate human monocytes via Toll-like receptor 4. Am J Respir Crit Care Med. 2013;187:1324–34. doi: 10.1164/rccm.201209-1602OC. [DOI] [PubMed] [Google Scholar]
  • 52.Shao MX, Nadel JA. Neutrophil elastase induces MUC5AC mucin production in human airway epithelial cells via a cascade involving protein kinase C, reactive oxygen species, and TNF-α-converting enzyme. J Immunol. 2005;175:4009–16. doi: 10.4049/jimmunol.175.6.4009. [DOI] [PubMed] [Google Scholar]
  • 53.Cockayne DA, Cheng DT, Waschki B, et al. Systemic biomarkers of neutrophilic inflammation, tissue injury and repair in COPD patients with differing levels of disease severity. PLoS ONE. 2012;7:e38629. doi: 10.1371/journal.pone.0038629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Bergin DA, Greene CM, Sterchi EE, et al. Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway. J Biol Chem. 2008;283:31736–44. doi: 10.1074/jbc.M803732200. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Induction of MUC5AC secretion by S100 protein in normal human bronchial epithelial cells.

imm0144-0079-sd1.pdf (10.3KB, pdf)

Figure S2. Induction of MUC5AC protein by S100A8 in normal human bronchial epithelial cells.

imm0144-0079-sd2.pdf (20.4KB, pdf)

Figure S3. The expression of TLR4 and RAGE mRNAs in airway epithelial cells.

imm0144-0079-sd3.pdf (31.8KB, pdf)

Figure S4. Effect of anti-receptor for advanced glycation end-products (RAGE) antibody on S100A12-mediated activation of extracellular signal-regulated kinase.

imm0144-0079-sd4.pdf (36.2KB, pdf)
imm0144-0079-sd5.doc (32KB, doc)

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES