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Published ahead of print on May 14, 2003, doi:10.1165/rcmb.2002-0286OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 523-530, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0286OC

Myeloid Related Protein-8/14 Stimulates Interleukin-8 Production in Airway Epithelial Cells

Ali Ahmad, Darren L. Bayley, Shiping He and Robert A. Stockley

Respiratory Research Laboratory, Department of Medicine, University of Birmingham, United Kingdom

Address correspondence to: Ali Ahmad, Department of Medicine, University of Birmingham, Birmingham B15 2TT, UK. E-mail: a.ahmad{at}bham.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Excessive neutrophil recruitment is implicated in the pathogenesis of chronic lung diseases by causing collateral tissue damage. The cells move from the circulation in response to chemokines, such as interleukin (IL)-8, that are secreted by several lung cell types including epithelial cells. This study has investigated factors present in bronchial secretions that are responsible for IL-8 expression and secretion by epithelial cells and hence initiate or perpetuate the recruitment of neutrophils. A549 epithelial cells were stimulated with proinflammatory molecules likely to be of relevance in the lung. Tumor necrosis factor-{alpha}, IL-1ß, and lipopolysaccharide stimulated IL-8 production from epithelial cells in a dose- and time-dependent manner, and these effects were abrogated by specific antibodies or inhibitors. Bronchial secretions also stimulated IL-8 production, and lipopolysaccharide accounted for ~ 33% of this activity. An abundant 32-kD protein capable of stimulating IL-8 production was isolated from the secretion and identified as neutrophil cytoplasmic protein myeloid-related protein (MRP)-14, which is the heavy polypeptide chain in the MRP-8/14 heterodimer. Abrogation of MRP-14 activity with a specific antibody also reduced the IL-8–stimulating potential of bronchial secretions, suggesting it was a significant stimulus to IL-8 production in the lung and may amplify the neutrophilic inflammation seen in bronchial disease.

Abbreviations: bovine serum albumin, BSA • enzyme-linked immunosorbent assay, ELISA • interleukin, IL • LPS-binding protein, LBP • lipopolysaccharide, LPS • myeloid-related protein, MRP • normal human bronchial epithelial cells, NHBE cells • polyvinylidene difluoride, PVDF • sodium dodecyl sulfate, SDS • saline sodium citrate, SSC • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic lung disease is a major worldwide cause of morbidity and mortality. Despite this, there are at present no specific treatments for diseases such as chronic bronchitis, bronchiectasis, chronic obstructive pulmonary disease, or emphysema. These conditions are thought to develop (at least in part) as a result of excessive neutrophilic lung inflammation (1), which is often initiated after exposure to factors such as cigarette smoke, pollution, and infection. The resulting inflammatory cascade includes the release of neutrophil chemokines such as interleukin (IL)-8. Neutrophil recruitment can partially amplify this process by further release of IL-8 (2).

The release of IL-8 by bronchial epithelial cells or cell lines has been demonstrated in response to a number of inflammatory stimuli, including cigarette smoke (3), bacterial endotoxin (4), proinflammatory cytokines such as tumor necrosis factor (TNF)-{alpha} and IL-1ß (5), and neutrophil elastase (6). IL-8 stimulates neutrophil adherence to unstimulated endothelium and transendothelial migration (7), as well as enhancing neutrophil binding to endothelium and extracellular matrix by increasing expression of CD11b/CD18. In addition, IL-8 stimulates exocytosis of lysosomal enzymes, including myeloperoxidase and elastase from azurophil granules (8), and bioactive lipids, such as 5-hydroxytetraeinoic acid and LTB4 (9); and it increases oxygen radical production (10). Because neutrophil products can be damaging to the lung and the potential for IL-8 to initiate and amplify neutrophilic recruitment (11), the identification of key mediators that establish this process is of great therapeutic importance.

The current study was designed to identify the components responsible for IL-8 production in human bronchial secretions from patients with chronic pulmonary disease using airway epithelial cells. The characterization of these molecules may highlight specific targets for reducing IL-8 generation and subsequently decreasing neutrophil recruitment in chronic inflammation. This may provide alternative therapeutic strategies for individuals susceptible to chronic inflammatory conditions and prevent excessive lung damage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Cytokines
TNF-{alpha}, anti–TNF-{alpha}, IL-1ß, anti–IL-1ß, Escherichia coli lipopolysaccharide, and polymyxin B sulfate were purchased from Sigma (Poole, UK). Anti–myeloid-related protein (MRP)-8/14 was purchased from Serotec (Oxford, UK), and MRP-8/14 was supplied by Barbro Isaksen and Magne Fagerhol (Department of Immunology and Transfusion Medicine, Ullevål Hospital, Oslo, Norway) purified from human leucocytes. Recombinant MRP-8/14 heterodimer and monomeric MRP-14 was a gift from Walter J Chazin (Vanderbilt University, Nashville, TN) and reconstituted in 25 mM Tris-HCl, pH 7.4.

Tissue Culture
Transformed human alveolar epithelial cells, A549 cells (ATCC CCL-185) were grown in a 1:1 (vol:vol) mixture of Dulbecco's modified Eagle's medium and Nutrient mixture F-10 HAM supplemented with 1% (vol/vol) 200 mM L-glutamine (Sigma) and 10% (vol/vol) heat-inactivated fetal calf serum (Gibco, Paisley, UK). Primary normal human bronchial epithelial cells (NHBE cells; Cambrex Bio Science Ltd, Wokingham, UK) were grown in bronchial epithelial growth media supplemented with BEGM Singlequots (Cambrex). Cells were grown at 37°C in 5% CO2 in vented 25cm2 flasks (Costar, High Wycombe, UK) and subcultured in 3.8cm2 12-well tissue culture clusters (Costar). The experiments reported here were performed using confluent cells in serum-free media (or unsupplemented media in the case of NHBE cells). Cell counts were determined by viewing detached cells on a haemocytometer at x400 magnification, and cell viability was determined by trypan blue exclusion.

IL-8 Assessment
The Quantikine human IL-8 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Abingdon, UK) was used to detect and quantify IL-8 secreted by the bronchial epithelial cells in culture. The kit has been tested stringently and validated for the measurement of IL-8 in complex biological fluids (12). The lower limit of detection was 10 pg/ml, and the inter-assay coefficient of variation was 6.73% (n = 6).

RNA Extraction
RNase contamination was minimized by pretreatment of plasticware and reagents with 0.1% (vol/vol) diethylpyrocarbonate (DEPC; Sigma) overnight, followed by autoclaving for 40 min at 121°C. A single-step method for RNA isolation by acid guanidinium isothiocyanate-phenol-chloroform extraction was adopted (13). RNA integrity was confirmed by agarose gel electrophoresis. RNA samples were denatured by addition of an equal volume of saline sodium citrate (SSC) (0.88% [wt/vol] NaCl/0.44% [wt/vol] sodium citrate): 37% formaldehyde (30:1) and heated to 60°C for 15 min. The RNA samples were loaded in duplicate onto a slot blot manifold and were bound to Hybond-N+ nylon membrane (Amersham Biosciences, Chalfort St. Giles, UK) under vacuum for 30 min. RNA-bound membranes were ultraviolet-fixed using an XL-1000 Spectrolinker and stored in the dark.

[32P]–IL-8 Hybridization
A Prime-a-Gene labeling kit (Promega, Southampton, UK) was used to specifically radiolabel purified IL-8 or ß-actin DNA. IL-8 or ß-actin cDNA was generated from the double-stranded template using the Klenow fragment of DNA polymerase I in a reaction mixture containing [{alpha}32P] dCTP (Amersham). The reaction mixture was incubated at room temperature for 60 min, and the reaction terminated by heating to 95°C for 2 min, cooling on ice, and adding 1 µmol EDTA. Pre-hybridization solution consisting of 6x SSC, 0.5% (wt/vol) sodium dodecyl sulfate (SDS), 5% (wt/vol) dextran sulfate, 5x Denhardt's solution (0.1% Ficoll type 400 [Amersham]/0.1% polyvinylpyrrolidone [Sigma]/ 0.1% bovine serum albumin [BSA]) was heated to 60°C. Mechanically sheared salmon sperm DNA (Sigma) was denatured at 95°C for 2 min and added (10 µl/ml) to the prehybridization solution. Northern or slot blots were saturated in 3x SSC placed in 20 ml prehybridization solution and incubated for 1 h at 65°C in a hybridization oven. The radiolabeled probe (10 µl) was added to the prehybridization solution and incubated overnight. The blots were serially washed with 2x to 0.5x SSC/0.5% (wt/vol) SDS for 30 min at 65°C. The blots were probed sequentially for IL-8 and ß-actin. After the first hybridization, the blot was stripped of radiolabeled DNA by overnight incubation at 65°C in the presence of 0.5% SDS. All specific IL-8 results were expressed in relation to the appropriate ß-actin units.

Abrogation of the Effects of Inflammatory Proteins and Lipopolysaccharide
Monoclonal antibodies to TNF-{alpha}, IL-1ß (Sigma), and MRP-8/14 (Serotec) were used to inhibit the effects of these proteins. Lipopolysaccharide (LPS) was inhibited by preincubation with polymyxin B sulfate bound to agarose beads (Sigma). The LPS-free solution was recovered by centrifugation at 200 x g.

Gel-Filtration Chromatography
Sephacryl S-100 (Sigma) expanded in excess phosphate-buffered saline (PBS) was applied to a 1.25-m gel filtration column to a produce a 250-ml gel bed volume. The elution buffer (PBS) was fed gravimetrically to the column inlet and the matrix washed with 5 vols (1.25 liter) elution buffer. A column flow rate of 20 ml/h was established for the collection of 7-ml eluate fractions at 4°C. A molecular weight marker kit (Sigma) was used to calibrate the column and each marker was loaded individually onto the column to prevent interaction between the proteins. Purulent sputum (known to contain a large number of neutrophils) was collected from a patient colonized with Branhamella catharalis (109 colony forming units/ml). The sputum was centrifuged at 50,000 x g for 90 min at 4°C to separate the fluid (sol phase) from the gel and cellular component. The purulent sol phase sputum sample (5 ml) was loaded onto the matrix and the optical density of each eluted fraction was determined by spectrophotometry at 280 nm.

SDS-Polyacrylamide Gel Electrophoresis
Molecules separated by gel-filtration chromatography were further resolved using SDS-polyacrylamide gel electrophoresis. A 12.5% resolving gel was prepared by combining 6.25 ml 29.1% (wt/vol) acrylamide/0.9% (wt/vol) bis-acrylamide with 3 ml running buffer (1.875 M Tris/HCl; pH 8.8), 150 µl 10% (wt/vol) SDS, 5.55 ml H2O, 50 µl 10% (wt/vol) ammonium persulphate, and 7.5 µl N,N,N',N'-tetramethylethylenediamine (TEMED; Sigma). The stacking gel consisted of 800 µl acrylamide/bis-acrylamide with 500 µl stacking buffer (1.25 M Tris/HCl; pH 6.8), 50 µl 10% (wt/vol) SDS, 3.6 ml H2O, 17 µl 10% (wt/vol) ammonium persulphate, and 5 µl TEMED. The electrophoresis buffer consisted of 25 mM Tris/192 mM glycine/0.1% (wt/vol) SDS. The gel filtration fractions were denatured in sample buffer (0.125 M Tris/HCl; pH 6.8 / 4% [wt/vol] SDS/20% [vol/vol] glycerol/10% [vol/vol] ß-mercaptoethanol / 2% [wt/vol] bromophenol blue) before PAGE. Protein bands were visualized after electrophoresis using silver stain (Sigma)

Polyvinylidene Difluoride Membrane Blotting
After electrophoresis, resolved protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane (Sigma) in blotting buffer (25 mM Tris/192 mM glycine/0.1% [wt/vol] SDS/ 20% [vol/vol] methanol) and electroblotted overnight at 100 mA in a gel blotter. Efficient transfer of proteins was confirmed by staining with brilliant blue G: methanol (1:1; Sigma). Immobilized protein bands were N-terminal sequenced by Alta Bioscience (Birmingham, UK), and amino acid sequences were identified from Swiss-Prot using an online database (14).

Statistical Analysis
All data was preanalyzed using the Shapiro-Wilk test (SPSS v.9) and shown to be normally distributed. A Student's t test for paired means was used to test for differences, and P values of < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-8 Production in Response to Classical Inflammatory Mediators and their Abrogation
TNF-{alpha}. Confluent A549 cells in serum-free media were stimulated with TNF-{alpha} ranging from 1–100 ng/ml for 24 h (Figure 1A). There was a 17-fold increase in IL-8 production detected in the supernatant of cells stimulated with 1 ng/ml TNF-{alpha} compared with unstimulated control cells, and this increased in a dose-dependent manner a further 3-fold at both 10 ng/ml and again at 100 ng/ml. IL-8 secretion into the supernatant of A549 cells was measured together with mRNA levels after stimulation by 10 ng/ml TNF-{alpha} at varying time intervals over 24 h (Figure 1B). IL-8 protein release in the incubation medium was detected after 2 h (1,539 ± 22 pg/106 cells). This doubled over the next 2 h (3,564 ± 360 pg/106 cells; P = 0.006) with a further 4-fold increase (13,289 ± 957 pg/106 cells; P < 0.001) occurring over the remaining 20 h of incubation. In contrast, elevated levels of IL-8 mRNA were detected after only 1 h (0.5 ± 0.02 units/ß-actin) and increased further by 2 h (P < 0.001). The amount of mRNA thereafter remained constant over the remaining 22 h of incubation. IL-8 stimulation by 10 ng/ml TNF-{alpha} was significantly reduced by the addition of 1 U/ml anti–TNF-{alpha} antibody (P = 0.001) and abolished by 10 U/ml (P < 0.0001; Figure 1C).



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  		Figure 1. IL-8 production in A549 cells in response to TNF-{alpha} for 24 h. (A) Effect of increasing concentration of TNF-{alpha}. (B) Time-dependent release of IL-8 and mRNA expression over 24 h after stimulation with 10 ng/ml TNF-{alpha}. Open bars, protein; line with diamonds, RNA. (C) Inhibition of IL-8 production stimulated by 10 ng/ml TNF-{alpha} following incubation with anti–TNF-{alpha} antibody. Data represent mean ± SEM, n = 4. *P <= 0.006 compared with control, **P < 0.001 compared with 1 h mRNA value.

 
IL-1ß. IL-1ß (ranging from 2.8 to 280U/ml) was incubated with A549 cells for 24 h (Figure 2A). An increase in IL-8 production of ~ 30 fold was detected in the supernatant of cells incubated with 2.8 U/ml IL-1ß compared with unstimulated cells (0.42 ± 0.04 ng/106 cells; P = 0.0003), and a further increase was observed at 28 U/ml (21.8 ± 2.1 ng/106 cells; P = 0.006). This effect was reduced by 35% by anti–IL-1ß antibody (1 U/ml) and abolished by 10 U/ml antibody (Figure 2B).



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  		Figure 2. IL-8 production from A549 cells in response to IL-1ß for 24 h. (A) Effect of increasing concentration of IL-1ß. (B) Inhibition of 28 U/ml IL-1ß with increasing concentration of anti–IL-1ß. Data represent mean ± SEM, n = 4. *P <= 0.006 compared with control.

 
LPS. A549 epithelial cells were stimulated with increasing concentrations of LPS (6.25–50 µg/ml) for 24 h. Control production over 24 h (133 ± 12.5 pg/106 cells) was increased 2.5-fold to 316 ± 47.8 pg/106 cells in response to 6.25 µg/ml LPS (P < 0.01). Increasing concentrations produced a dose-dependent effect rising to 504 ± 26.8/106 cells at 50 µg/ml (Figure 3A).



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  		Figure 3. IL-8 production from A549 cells in response to E. coli LPS over 24 h. (A) Effect of increasing concentration of LPS. (B) Abolition of the endotoxin effect of 50 µg/ml LPS following treatment with polymyxin-linked agarose beads. BSA-linked agarose beads were used as a control to show abrogation was specifically polymyxin B–related. Data represent mean ± SEM, n = 4. *P <= 0.01 compared with control.

 
The stimulatory effect of LPS was inhibited by removing the LPS from the culture media with polymyxin B–coated agarose beads (Figure 3B).

IL-8 Production in Response to Neutrophil Elastase
In the experiments performed here neutrophil elastase failed to stimulate IL-8 protein secretion and mRNA expression by A549 cells. A similar negative result was obtained with untransformed epithelial cells and primary bronchial epithelial cells (data not shown), suggesting that this enzyme is unlikely to be bioactive in relation to IL-8 production in the lung. The activity of elastase was confirmed using a specific substrate (N-succinyl-leu-(ala)3-p-nitroanilide), and was also shown to be active by stimulating secretory leukoprotease inhibitor mRNA expression in A549 cells (data not shown).

IL-8 Production in Response to Bronchial Secretions
Spontaneous sputum samples were pooled from six further patients and treated as described in MATERIALS AND METHODS. The pooled sol phase sputum was diluted in serum-free cell culture media and sterile filtered before addition to bronchial epithelial cells. The sol phase solutions were added to confluent A549 cells in doubling amounts ranging from 6.25–25% (vol/vol), and IL-8 release was measured after a 24 h incubation (Figure 4A). IL-8 production was stimulated in cells incubated with 6.25% (vol/vol) secretion from a control value of 0.44 ± 0.01 to 11.6 ± 0.49 ng/106 cells (P < 0.05) and concentrations were increased up to 3-fold (26.8 ± 0.9 ng/106 cells; P = 0.0005) in cells stimulated with 25% (vol/vol) sol phase solution. IL-8 measured in response to 5% (vol/vol) bronchial secretion over time (Figure 4B) showed that IL-8 was detected in the supernatant of stimulated cells after 1 h (387 ± 16 pg/106 cells), and the concentration increased to 924 ± 64 pg/106 cells over the next hour (P = 0.001). IL-8 concentration continued to increase after 4 h and 8 h (P < 0.005), and was elevated 35-fold (compared with the 1-h value) after 24 h to a total concentration of 13,678 ± 1,309 pg/106 cells (P < 0.0001).




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  		Figure 4. IL-8 production from A549 cells in response to bronchial secretions over 24 h. (A) Effect of increasing concentration of sputum sol phase. (B) Time-dependent release of IL-8 in response to 5% (vol/vol) sol phase. (C) Effect of increasing concentrations of anti–TNF-{alpha} antibody. (D) Effect of increasing concentrations of anti-IL-1ß antibody. (E) Contribution of LPS by removal of the endotoxin component using polymyxin-linked agarose beads, the effect of inert BSA-linked beads is also shown. Data represent mean ± SEM, n = 6. *P <= 0.03 compared with control values.

 
Contribution of Putative Stimulators of IL-8
The contribution of any TNF-{alpha} or IL-1ß present in lung secretions to IL-8 production by epithelial cells was investigated by the use of specific antibodies. No reduction in IL-8 production was observed in cells incubated with up to 1 U/ml anti–TNF-{alpha} (Figure 4C) or to 10 U/ml anti–IL-1ß antibody (Figure 4D). The endotoxin component in bronchial secretions was removed by incubating sol phase sputum with polymyxin B–coated agarose beads (2 mg agarose beads per ml media) overnight at 4°C and then sterile-filtered. IL-8 production induced by sputum sol phase was significantly reduced (P = 0.001) from 5,208 ± 529 to 2,860 ± 346pg/106 cells over 24 h compared with a control (media alone) of 557 ± 16 pg/106 cells. However, cells incubated with BSA-linked agarose alone also showed a decrease in IL-8 production from 5,208 ± 529 to 4,257 ± 215 pg/106 cells (P = 0.003). This was not due to degradation of sputum components during the overnight incubation, as the sputum control was treated in the same way. The results therefore suggest that the BSA or agarose may have "nonspecifically" bound or retarded one or more sputum components that reduced the positive effect on IL-8 production. When this "nonspecific" effect was taken into account, the results suggest that the specific contribution of LPS in IL-8 production by sputum was ~ 30% (Figure 4E).

Fractionation of Sputum Sol Phase
Purulent sputum sol phase was fractionated by gel filtration chromatography through Sephacryl S-100. The absorbance of each eluted fraction was determined at 280 nm, and is shown as an elution profile in Figure 5A. Two major peaks of protein were observed in fractions corresponding to molecular weights of 135 and 55 kD, respectively.




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  		Figure 5. IL-8 production in A549 cells in response to fractionated sputum sol phase. (A) Absorbance profile of eluted fractions. (B) IL-8 production profile in response to 5% (vol/vol) eluted fractions. Molecular weight of marker proteins is indicated. (C) IL-8 production in response to 6 µg/ml eluted fractions. Approximate molecular weight is indicated. (D) Effect of 1 U/ml anti–TNF-{alpha} and 10 U/ml anti–IL-1ß antibodies in cells incubated for 24 h with the 32-kD fraction (48) and the 14-kD fraction (56). Open bar, stimulus alone; checkered bar, anti-TNF; filled bar, anti-IL-1. (E) An SDS-PAGE gel (silver stained) of sputum sol phase (fraction numbers: 45–51), the predominant protein band (MRP-14) at 14 kD is indicated.

 
The sputum sol-phase fractions obtained by gel filtration chromatography were sterile-filtered and diluted to 5% (vol/vol) in serum-free media. Fractions 25–60 were then incubated with confluent A549 cells for 24 h, and the IL-8 secreted was measured by ELISA. Increased secretion of IL-8 was observed from cells incubated with fractions 27–58, but the major peaks were produced by fractions 42 and 48, corresponding to molecular weights of ~ 62 and 32 kD, respectively (Figure 5B).

Because the protein content of each fraction varied, the relative potency of each fraction was assessed based on the absorbance values used to calculate the protein concentration (assuming that an optical density of 1.00 was equivalent to 1 mg/ml protein). IL-8 production was then reassessed following incubation of A549 cells for 24 h with an estimated 6 µg protein/ml using sputum fractions 33–57 for 24 h (Figure 5C). In contrast to the results obtained by stimulating cells with fractions at a fixed volume, four major peaks of activity were identified that stimulated IL-8 secretion. These peaks of activity corresponded to protein molecules of ~ 150, 69, 25, and 14 kD. The production of IL-8 stimulated by these fractions including fractions 48 or 56 (equivalent to a molecular weight of 36 and 14 kD, respectively) was not inhibited by antibodies to TNF-{alpha} or IL-1ß (Figure 5D).

SDS-PAGE
PAGE of fraction 48 (the most potent fraction vol:vol, Figure 5B) through a nondenaturing 7.5% acrylamide gel produced a single band corresponding to ~ 29 kD (data not shown). SDS-PAGE of fraction 48 produced an intense band of ~ 14 kD (Figure 5E), which was electroblotted onto PVDF membrane, and the sequence was determined. The sequence found (Met Leu Thr Glu Leu Glu Lys Ala Leu Asn Ser Ile Ile Asp Val) showed complete amino acid identity with the neutrophil cytoplasmic protein MRP-14, suggesting this was the major constituent of fraction 48.

IL-8 Production in Response to MRP-8/14
A549 cells incubated with 10 and 15 µg/ml purified MRP-8/14 showed a significant increase in IL-8 production over 24 h from a control value of 721 ± 21 to 1,680 ± 6,820 pg/106 cells (P < 0.001; Figure 6A). This was accompanied by an increase in IL-8 mRNA expression from undetectable levels to 135 ± 3 U/ß-actin. There was no further increase in IL-8 production in response to MRP-8/14 at concentrations of up to 125 µg/ml. The stimulatory effect of MRP-8/14 was inhibited ~ 65% (accounting for the unstimulated control value of 651 ± 15 pg/106 cells) by anti–MRP-8/14 antibody from a stimulated value of 1,365 ± 110 to 901 ± 84 pg/106 cells (P < 0.005; Figure 6C). MRP-8/14 stimulated a time-dependent release of IL-8, with detectable levels after 1 h that continued to increase up to 24 h (1,524 ± 63 pg/106 cells; Figure 6B). Almost maximal IL-8 mRNA expression response was detected after 1 h (140 ± 1 U/ß-actin) and remained essentially constant throughout the time course.




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  		Figure 6. IL-8 production in airway epithelial cells stimulated by MRP-8/14. (A) IL-8 release and mRNA expression in response to MRP-8/14 (calprotectin). (B) Time-dependent release and mRNA expression of IL-8 in response to 15 µg/ml MRP-8/14. (C) Inhibitory effect of increasing concentrations of anti-MRP-8/14 (anti-calprotectin) antibody in cells stimulated by 15 µg/ml MRP-8/14. (D) Abrogation of sol phase–induced IL-8 release and mRNA expression by 10 U/ml anti–MRP-8/14 (anti-calprotectin) antibody. (E) Effect of 15 µg/ml MRP-8/14 and MRP-14 alone in primary bronchial epithelial cells. Tris buffer diluted equivalently in media is shown for comparison together with media alone. Data represent mean ± SEM, n = 6. *P <= 0.01 compared with control values: **P <= 0.05 compared with sputum value. A, B, and D: open bars, protein; lines with diamonds, RNA.

 
The contribution of MRP-8/14 to the sol-phase sputum effect was tested using the anti–MRP-8/14 antibody (Figure 6D). The sol phase increased IL-8 production from a control value of 577 ± 34 (media alone) to 1,707 ± 76 pg/106 cells. In the presence of 10 U/ml antibody, this increase was reduced to 1,395 ± 65 (P = 0.02), suggesting that MRP-8/14 accounted for 28% of the effect and was accompanied by a reduction in mRNA expression (from 190 ± 9 to 166 ± 7 U/ß-actin). MRP-8/14 activity could not be abrogated using Zn2+ as reported elsewhere (15) as 10–100 µM Zn2+ itself stimulated IL-8 release (data not shown).

Both the MRP-8/14 heterodimer and monomeric MRP-14 stimulated IL-8 production in NHBE cells (Figure 6E). Supernatant IL-8 levels increased to 2,760 ± 192 pg/106 cells from a control value of 225 ± 20 pg106 cells (P < 0.001) when stimulated with 15 µg/ml MRP-8/14, whereas an increase to 2,229 ± 283 pg/106 cells was observed with a stimulus of 15 µg/ml MRP-14 (P < 0.001).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Fractionation of sputum sol phase revealed potent IL-8–stimulating factors corresponding to molecular weights of 14, 25, 69, and 150 kD. The most prominent stimulatory fraction (based on volume) was equivalent to a molecular size of 32 kD, whereas the most potent fraction when corrected for protein concentration was ~ 14 kD. Further investigation confirmed that these factors were neither TNF-{alpha} nor IL-1ß. SDS-PAGE and subsequent N-terminal sequencing of the eluted band identified MRP-14 as the major constituent in the 32-kD fraction.

MRP-8/14 comprises 60% of the neutrophil cytosolic protein (16) and belongs to the S100 family of proteins (10–14 kD), some of which have been reported to act as chemoattractants. S100A2 is thought to be chemotactic for eosinophils (17) and S100A7 for neutrophils and CD4+ T lymphocytes (18). MRP-8/14 has been shown to bind endothelial cells in conjunction with sulphated glycosaminoglycan structures (19) or {alpha}2-macroglobulin (20). MRP-8/14 is a noncovalently associated trimer or dimer consisting of a combination of the 11-kD light L1 (L1l, S100A8) and 14.5-kD heavy L1 (L1h, MRP-14, S100A9) polypeptide chains each with two calcium-binding domains. The 14-kD protein observed in the experiments described here corresponded to the MRP-14 subunit of MRP-8/14, whereas the undenatured 32-kD protein most likely corresponded to a MRP-8/14 trimer consisting of two MRP-8 and one MRP-14 subunits.

MRP-8/14 levels in neutrophils have been shown to rise rapidly during the onset of fever and high plasma MRP-8/14 levels have been found in patients with cystic fibrosis (21) and in the synovial fluid of patients with rheumatoid arthritis (22). Elevated levels of plasma and airway secretion MRP-8/14 indicative of neutrophil turnover have also been associated with chronic obstructive bronchitis, fibrosing alveolitis, and cystic fibrosis. These levels were shown to be reversible, falling after treatment of the inflammation by antibiotics or steroids (23). The physiologic role of MRP-8/14 is not well characterized; however, it is known to be apoptotic for tumor cells (24), may inhibit tumor invasiveness through matrix metalloprotease inhibition (15), and it is antimicrobial (25) possibly through sequestration of Zn2+ and MRP-8/14–expressing epithelial cells show reduced bacterial binding and internalisation (26). Although MRP-14 activates CD11b/CD18 (Mac-1), thereby facilitating endothelial adhesion via intercellular adhesion moelcule-1, it is not directly chemotactic for neutrophils and does not trigger neutrophil activation such as superoxide anion generation, cytoskeletal remodeling, or an increase in intracellular Ca2+ (27). However, MRP-8/14–induced epithelial IL-8 secretion may play a crucial role in the amplification of neutrophil migration in to the lung by a paracrine mechanism. This suggests a unique role for this protein in airways inflammation in chronic lung disease.

Studies with purified MRP-8/14 confirmed the ability of this protein to stimulate IL-8 production by A549 cells acting at the transcriptional level. The monoclonal antibody was able to abrogate this effect, although not completely. This may be because the monoclonal antibody is epitope-specific and may incompletely cover the active region of one of the subunits, or alternatively the subunits may act independently in this respect. However, similar studies with the pooled bronchial secretions demonstrated that at least 28% of the IL-8–stimulating potential was dependent on MRP-8/14.

Both recombinant heterodimeric MRP-8/14 and monomeric MRP-14 stimulated IL-8 release from primary bronchial epithelial cells, suggesting that this effect was not restricted to the A549 cell line and may therefore be relevant to inflammation at both the bronchial and alveolar region of the lung. Although Newton and coworkers (27) showed that the functional effect (activation of neutrophil CD11b/CD18) of MRP-14 was abolished in the presence of the MRP-8 subunit, no such inhibitory effect was observed in the experiments reported here. Furthermore, the response to the heterodimer is likely to be clinically relevant, as free MRP-14 has not been observed in vivo. In addition, a stable heterodimer is necessary for the MRP-8/14–mediated antimicrobial function because the zinc-binding domain is thought to involve residues on both polypeptide chains (28). The role of the individual subunits or multimers, and their contribution in individual patients with chronic lung disease, remains to be determined. Nevertheless, the studies reported here suggest that MRP-8/14 may be of more importance than conventional cytokines in the amplification of lung inflammation.

TNF-{alpha} was shown to induce IL-8 in a time- and dose-dependent manner from epithelial cells, corroborating evidence from other studies (29), and this effect was abrogated by anti–TNF-{alpha} antibody. A similar time- and dose-dependent release of IL-8 was stimulated by IL-1ß and LPS, and this effect was also abolished using specific antibody and polymyxin B, respectively. Interestingly, LPS elicited this response in A549 cells in the absence of endogenous lipopolysaccharide-binding protein (LBP) which, together with LPS, interacts with CD14 on the cell surface, leading to intracellular signaling (30). LPS stimulation in the experiments reported here may have acted in an LBP-independent manner by binding directly to CD14 on the epithelial surface, because although A549 cells produce LBP in response to IL-1, TNF-{alpha}, and IL-6, this has not been reported in response to LPS (31).

IL-8 is believed to be responsible for the accumulation of neutrophils in the airways and is a major determinant of neutrophil chemotaxis in response to sputum from patients with chronic lung disease, both in the stable state and during exacerbations (32). Thus, the levels of IL-8 in these secretions will influence neutrophil influx and hence the severity of inflammation. Sol phase of sputum was shown to induce a time- and dose-dependent secretion of IL-8 by epithelial cells that was comparable to the response achieved using potent IL-8 inducers such as IL-1ß or TNF-{alpha}.

Studies have shown that TNF-{alpha} is present in bronchial secretions (33), and its production may potentially be induced by LPS in the secretion (34). However, a significant TNF-{alpha}–mediated effect on IL-8 production was not detected in the sputum samples in the present study by use of a monoclonal antibody to abrogate any effect. Similar methodology indicated that IL-1ß was not responsible for IL-8 production by the lung secretions.

Any endotoxin component responsible for induction of IL-8 in sol phase sputum was tested by affinity chromatography with polymyxin B–linked agarose beads. Although a BSA-linked agarose control also abrogated some sputum factor(s) that induced IL-8 production, the reduction of IL-8 production by removal of endotoxin from sputum was significantly greater than that observed in response to BSA alone (P < 0.001). On the basis of these experiments, it can be concluded that LPS accounted for ~ 30% of the stimulatory effect of sputum on IL-8 production by epithelial cells.

Neutrophil elastase is also often present in lung secretions (35), and has been implicated in IL-8 production. However, in our experiments elastase did not activate IL-8 production from A549 cells, untransformed bronchial epithelial cells, or primary bronchial epithelial cells. The absence of any effect was not related to cell death, cell detachment, a false negative result due to interference with the ELISA, or due to enzyme inactivity. In contrast to our findings, previous studies have suggested that IL-8 production is increased by elastase. An SV40-transformed epithelial cell line (6) was used, however, suggesting that this may be a cell line–specific phenomenon.

In summary, the evidence presented here has identified two factors, endotoxin (which is bacterial in origin) and MRP-8/14 (which is host-derived), that are major constituents in the secretions capable of stimulating IL-8 production by lung airway cells. The data confirm a hitherto unknown role for MRP-8/14 in inflammation, acting as a potential amplification factor. The exact mechanism remains unknown at present, but targeting this protein or its effects may provide new therapeutic strategies that can modulate neutrophilic inflammation. Such approaches may be of major importance in reducing collateral damage in diseases as diverse as rheumatoid arthritis and chronic lung diseases such as cystic fibrosis and emphysema.


   Acknowledgments
 
This work was funded by research grants from the Endowment Fund of the United Birmingham Hospitals and Aventis Pharma S.A. Prof. Magne Fagerhol, Dr. Barbro Isaksen, and Prof. Walter Chazin were most helpful in discussing the roles of MRP-8/14, and provided the purified and recombinant molecules. Dr. Natalia Carrabino provided much support in the culture of primary cells. The assistance and feedback from Dr. Sarah Nuttall was highly appreciated.

Received in original form December 3, 2002

Received in final form April 17, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Stockley, R. A. 1995. The pathogenesis of chronic obstructive lung diseases: implications for therapy. QJM 88:141–146.
  2. Cassatella, M. A., I. Guasparri, M. Ceska, F. Bazzoni, and F. Rossi. 1993. Interferon-gamma inhibits interleukin-8 production by human polymorphonuclear leucocytes. Immunology 78:177–184.[Medline]
  3. Shoji, S., R. F. Ertl, and S. Koyama. 1995. Cigarette smoke stimulates release of neutrophil chemotactic activity from cultured bovine epithelial cells. Clin. Sci. 88:337–344.[Medline]
  4. Koyama, S., S. I. Rennard, and G. Leikauf. 1991. Endotoxin stimulates bronchial epithelial cells to release chemotactic factors for neutrophils. J. Immunol. 147:4293–4301.[Abstract]
  5. Standiford, T. J., S. L. Kunkel, M. A. Basha, S. W. Chensue, J. P. d. Lynch, G. B. Toews, J. Westwick, and R. M. Strieter. 1990. Interleukin-8 gene expression by a pulmonary epithelial cell line: a model for cytokine networks in the lung. J. Clin. Invest. 86:1945–1953.
  6. Nakamura, H., K. Yoshimura, N. G. McElvaney, and R. G. Crystal. 1992. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line. J. Clin. Invest. 89:1478–1484.
  7. Baggiolini, M. 1995. Activation and recruitment of neutrophil leukocytes. Clin. Exp. Immunol. 101:5–6.
  8. Djeu, J. Y., K. Matsushima, J. J. Oppenheim, K. Shiotsuki, and D. K. Blanchard. 1990. Functional activation of human neutrophils by recombinant monocyte-derived neutrophil chemotactic factor/IL-8. J. Immunol. 144:2205–2210.[Abstract]
  9. Schroeder, J.-M. 1989. The monocyte-derived neutrophil activating peptide (NAP-1/interleukin 8) stimulates human neutrophil arachidonate-5-lipoxygenase, but not the release of cellular arachidonate. J. Exp. Med. 170:847–863.[Abstract/Free Full Text]
  10. Schroeder, J.-M., U. Mrowietz, E. Morita, and E. Christophers. 1987. Purification and partial characterization of a human monocyte-derived neutrophil activating peptide. J. Immunol. 139:3474–3483.[Abstract]
  11. Gompertz, S., C. O'Brien, D. L. Bayley, S. L. Hill, and R. A. Stockley. 2001. Changes in bronchial inflammation during acute exacerbations of chronic bronchitis. Eur. Respir. J. 17:1112–1119.[Abstract/Free Full Text]
  12. Stockley, R. A., and D. L. Bayley. 2000. Validation of assays for inflammatory mediators in sputum. Eur. Respir. J. 15:778–781.[Abstract]
  13. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159.[Medline]
  14. Pearson, W. R., and D. J. Lipman. 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85:2444–2448.[Abstract/Free Full Text]
  15. Isaksen, B., and M. K. Fagerhol. 2001. Calprotectin inhibits matrix metalloproteinases by sequestration of zinc. Mol. Pathol. 54:289–292.[Abstract/Free Full Text]
  16. Dale, I., P. Brandtzaeg, M. K. Fagerhol, and H. Scott. 1985. Distribution of a new myelomonocytic antigen (L1) in human peripheral blood leukocytes: immunofluorescence and immunoperoxidase staining features in comparison with lysozyme and lactoferrin. Am. J. Clin. Pathol. 84:24–34.[Medline]
  17. Komada, T., R. Araki, K. Nakatani, I. Yada, M. Naka, and T. Tanaka. 1996. Novel specific chemtactic receptor for S100L protein on guinea pig eosinophils. Biochem. Biophys. Res. Commun. 220:871–874.[CrossRef][Medline]
  18. Jinquan, T., H. Vorum, C. G. Larsen, P. Madsen, H. H. Rasmussen, B. Gesser, M. Etzerodt, B. Honore, J. E. Celis, and K. Thestrup-Pedersen. 1996. Psoriasin: a novel chemotactic protein. J. Invest. Dermatol. 107:5–10.[CrossRef][Medline]
  19. Robinson, M. J., P. Tessier, R. Poulsom, and N. Hogg. 2002. The S100 family heterodimer, MRP-8/14, binds with high affinity to heparin and heparan sulfate glycosaminoglycans on endothelial cells. J. Biol. Chem. 277:3658–3665.[Abstract/Free Full Text]
  20. Eue, I., S. Konig, J. Pior, and C. Sorg. 2002. S100A8, S100A9 and the S100A8/A9 heterodimer complex specifically bind to human endothelial cells: identification and characterization of ligands for the myeloid-related proteins S100A9 and S100A8/A9 on human dermal microvascular endothelial cell line-1 cells. Int. Immunol. 14:287–297.[Abstract/Free Full Text]
  21. Golden, B. E., P. A. Clohessy, G. Russell, and M. K. Fagerhol. 1996. Calprotectin as a marker of inflammation in cystic fibrosis. Arch. Dis. Child. 74:136–139.[Abstract]
  22. Berntzen, H. B., U. Olmez, M. K. Fagerhol, and E. Munthe. 1991. The leukocyte protein L1 in plasma and synovial fluid from patients with rheumatoid arthritis and osteoarthritis. Scand. J. Rheumatol. 20:74–82.[Medline]
  23. Stockley, R. A., I. Dale, S. L. Hill, and M. K. Fagerhol. 1984. Relationship of neutrophil cytoplasmic protein (L1) to acute and chronic lung disease. Scand. J. Clin. Lab. Invest. 44:629–634.[Medline]
  24. Yui, S., M. Mikami, and M. Yamazaki. 1995. Induction of apoptotic cell death in mouse lymphoma and human leukemia cell lines by a calcium-binding protein complex, calprotectin, derived from inflammatory peritoneal exudate cells. J. Leukoc. Biol. 58:650–658.[Abstract]
  25. Loomans, H. J., B. L. Hahn, Q. Q. Li, S. H. Phadnis, and P. G. Sohnle. 1998. Histidine-based zinc-binding sequences and the antimicrobial activity of calprotectin. J. Infect. Dis. 177:812–814.[Medline]
  26. Nisapakultorn, K., K. F. Ross, and M. C. Herzberg. 2001. Calprotectin expression inhibits bacterial binding to mucosal epithelial cells. Infect. Immun. 69:3692–3696.[Abstract/Free Full Text]
  27. Newton, R. A., and N. Hogg. 1998. The human S100 protein MRP-14 is a novel activator of the beta 2 integrin Mac-1 on neutrophils. J. Immunol. 160:1427–1435.[Abstract/Free Full Text]
  28. Sohnle, P. G., M. J. Hunter, B. Hahn, and W. J. Chazin. 2000. Zinc-reversible antimicrobial activity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and 14). J. Infect. Dis. 182:1272–1275.[CrossRef][Medline]
  29. Kwon, O. J., B. T. Au, P. D. Collins, I. M. Adcock, J. C. Mak, R. R. Robbins, K. F. Chung, and P. J. Barnes. 1994. Tumor necrosis factor-induced interleukin-8 expression in cultured human airway epithelial cells. Am. J. Physiol. 267:L398–L405.
  30. Wright, S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, and J. C. Mathison. 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249:1431–1433.[Abstract/Free Full Text]
  31. Dentener, M. A., A. C. Vreugdenhil, P. H. Hoet, J. H. Vernooy, F. H. Nieman, D. Heumann, Y. M. Janssen, W. A. Buurman, and E. F. Wouters. 2000. Production of the acute-phase protein lipopolysaccharide-binding protein by respiratory type II epithelial cells: implications for local defense to bacterial endotoxins. Am. J. Respir. Cell Mol. Biol. 23:146–153.[Abstract/Free Full Text]
  32. Mikami, M., C. G. Llewellyn-Jones, D. Bayley, S. L. Hill, and R. A. Stockley. 1998. The chemotactic activity of sputum from patients with bronchiectasis. Am. J. Respir. Crit. Care Med. 157:723–728.[Abstract/Free Full Text]
  33. Bonfield, T. L., J. R. Panuska, M. W. Konstan, K. A. Hilliard, J. B. Hilliard, H. Ghnaim, and M. Berger. 1995. Inflammatory cytokines in cystic fibrosis lungs. Am. J. Respir. Crit. Care Med. 152:2111–2118.[Abstract]
  34. DeForge, L. E., J. S. Kenney, M. L. Jones, J. S. Warren, and D. G. Remick. 1992. Biphasic production of IL-8 in lipopolysaccharide (LPS)-stimulated human whole blood: separation of LPS- and cytokine-stimulated components using anti-tumor necrosis factor and anti-IL-1 antibodies. J. Immunol. 148:2133–2141.[Abstract]
  35. Stockley, R. A., D. Bayley, S. L. Hill, A. T. Hill, S. Crooks, and E. J. Campbell. 2001. Assessment of airway neutrophils by sputum colour: correlation with airways inflammation. Thorax 56:366–372.[Abstract/Free Full Text]



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