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Published ahead of print on April 24, 2003, doi:10.1165/rcmb.2002-0306OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 483-489, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0306OC

Serum and Low-Density Lipoprotein Enhance Interleukin-8 Secretion by Airway Epithelial Cells

James E. Gern, Rebecca Brockman-Schneider, Saswati Bhattacharya, James S. Malter and William W. Busse

Departments of Pediatrics, Pathology, and Medicine, University of Wisconsin-Madison, Madison, Wisconsin

Address correspondence to: James E. Gern, M.D., K4/918 CSC, 600 Highland Avenue, Madison, WI 53792-9988. E-mail: gern{at}medicine.wisc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Viral respiratory infections rapidly increase vascular permeability, which leads to the transudation of serum proteins into airway secretions and tissues. To determine whether this process activates airway epithelial cells, bronchial epithelial cells were incubated with serum, and interleukin (IL)-8 secretion and gene expression were examined. As little as 0.1% serum significantly enhanced IL-8 secretion, and maximal secretion (65 ± 4 ng/ml, 48 h) was observed with 10% serum. Low-density lipoprotein, but not albumin or immunoglobulin G, augmented bronchial epithelial IL-8 secretion, which was partially blocked by a monoclonal antibody specific for the low-density lipoprotein receptor. The IL-8–inducing activity of plasma was also augmented by clotting and platelet activation. Mechanistically, serum activated nuclear factor-{kappa}B and increased the stability and steady state levels of IL-8 mRNA. In summary, specific components of serum are potent activators of IL-8 mRNA and secretion, and the increased IL-8 production is likely to be a result of both increased transcription and mRNA stability. This effect may represent an innate mechanism for the recruitment of neutrophils to the airway in response to noxious stimuli, such as viral infections, that increase vascular permeability.

Abbreviations: bronchial epithelial, BE • bronchial epithelial growth medium, BEGM • granulocyte-colony stimulating factor, G-CSF • immunoglobulin, Ig • interleukin, IL • low-density lipoprotein, LDL • nuclear factor-{kappa}B, NF-{kappa}B • rhinovirus, RV • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There is mounting evidence that the immune response to respiratory viral infections includes the generation of inflammation that contributes to the pathogenesis of respiratory symptoms and abnormal airway physiology. In fact, for rhinovirus (RV) infections, which infect only a small fraction of respiratory epithelial cells and do not cause appreciable cytopathic effects (1, 2), the immune response to the virus may be the major factor driving respiratory symptoms (3). Understanding mechanisms of virus-induced inflammation may therefore be the key to the development of new therapeutic approaches to respiratory viral infections. This goal is especially important for individuals with asthma, chronic obstructive pulmonary disease, or cystic fibrosis, who are at risk for exacerbations of chronic pulmonary diseases during infections with common cold viruses such as RV (4–6).

Rhinorrhea is one of the earliest symptoms of a viral upper respiratory infection. During the initial stage of a viral upper respiratory infection, nasal secretions contain increased concentrations of plasma proteins (e.g., albumin, immunoglobulin [Ig]G), indicating that the permeability of the nasal vasculature is increased (7). Moreover, peak levels of plasma proteins in nasal secretions coincide with the time of maximal cold symptoms (7) and virus-induced increases in bronchial responsiveness (8), suggesting the possibility that the influx of plasma proteins may contribute to airway dysfunction.

Following an initial increase in plasma proteins, the composition of nasal fluid changes to include relatively greater concentrations of proteins such as IgA and mucins that are secreted by the epithelium and goblet cells (7, 9). Concurrently, there is an increase in neutrophils in nasal secretions, together with cytokines (granulocyte-colony stimulating factor [G-CSF] and chemokines (e.g., interleukin [IL]-8) that regulate neutrophil synthesis, recruitment, and activation (10–12). The temporal sequence of these events suggests the possibility of a cause and effect relationship: the transudation of blood proteins, which occurs early during respiratory viral infections, may stimulate epithelial cells to secrete IL-8 and G-CSF, and thereby initiate or promote neutrophilic airway inflammation. To test this hypothesis, we have developed an in vitro model using primary cultures of nontransformed bronchial epithelial (BE) cells to determine the effects of serum or plasma proteins on IL-8 and G-CSF secretion, and the potential mechanisms for these effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Epithelial Cell Isolation and Culture
BE cells were obtained from residual surgical tissue trimmed from lungs during the process of transplantation, or from healthy tissue adjacent to resected lung tumors. The bronchial specimens were dissected and enzymatically digested, and monolayers of BE cells were isolated as previously described (13). The University of Wisconsin Human Subjects Committee approved the experimental protocol.

Cell monolayers were grown in 24-well tissue culture plates, and were maintained in bronchial epithelial growth medium (BEGM; BioWhittaker Inc., Walkersville, MD). For these experiments, the BEGM medium was replaced by medium (BEGM-4) lacking hydrocortisone, epithelial growth factor, epinephrine, and bovine pituitary extract, in an effort to remove factors that could influence IL-8 secretion.

In some experiments, a monoclonal antibody to the human low-density lipoprotein (LDL) receptor (C7) was used to block LDL attachment to epithelial cells. The hybridoma secreting the C7 antibody (Cat no CRL-1691) was obtained from American Type Culture Collection, Manassas, VA. The cells were injected intraperitoneally into Balb/c mice. Mice were watched carefully and were killed before harvest of the ascites fluid. The antibody was purified by affinity chromatography using HiTrap protein A-Seharose affinity columns (Amersham Biosciences, Piscataway, NJ). The antibody was used at a concentration of 50 µg/ml.

Serum and Plasma
For the majority of experiments, serum was prepared by collecting blood from healthy donors under sterile conditions using marble top vacutainer tubes with plugs. Collected blood was allowed to clot for 15–30 min, followed by separation of serum from the clot by centrifugation (2,000 rpm, 10 min).

To separate serum proteins on the basis of molecular weight, Serum was prefiltered through a 0.22-µ filter. After prerinsing filters (3, 10, 30, 50, 100, and 500 x 103 MW cutoff Centricon filters; Millipore, Billerica, MA) with sterile water, 1 ml of serum was added to each filter, and the assemblies were centrifuged (1 h at 1,000 x g for 30 and 100 K filters; 2 h at 5,000 x g for all others according to the manufacturer's guidelines). The filtrates were stored at 4°C for use in cell culture

For selected experiments, serum and plasma were prepared from blood collected in sodium citrate tubes (blue tops). Serum was procured by adding 120 µl of 1 M CaCl2 to the tube and allowing the blood to clot for 30 min followed by centrifugation (2,000 rpm, 10 min) to separate serum from the clotted material. Plasma was separated from blood collected in sodium citrate tubes simply by centrifugation (3,600 rpm, 10 min.). Collected plasma was then centrifuged further (15,000 rpm, 18 min.) to remove platelets. Half of this platelet-free plasma was reserved, and the remaining volume was treated with 1 M CaCl2 (30 µl/ml plasma) and allowed to clot. Subsequent centrifugation (2,000 rpm, 10 min.) separated platelet-free serum from the clotted material. Serum and plasma samples were stored at -20°C pending use in experiments.

In addition, some experiments used delipidated serum, and this was prepared using two different techniques. In the first method, Aerosil (fumed silica) was added to serum in the amount of 20 mg Aerosil/ml serum. Mixture was thoroughly vortexed and then allowed to mix overnight on a rocking platform. Serum was separated from Aerosil by centrifugation (2,000 x g, 15 min.), followed by treating the collected serum a second time with fresh Aerosil. A second method of delipidation involved organic extraction according to the method of Cham and Knowles (14). A given quantity of serum was extracted twice using this method to achieve removal of lipids.

LDL (catalog #L 7,914), purified from human plasma, was obtained from Sigma Inc. (St Louis, MO).

Cytokine Quantitation
Levels of IL-8, eotaxin, and G-CSF were measured by enzyme-linked immunosorbent assay. IL-8 was measured as previously described (13), and eotaxin and G-CSF were measured with commercially available kits (R&D Systems, Minneapolis, MN). The sensitivity of the assays are 6, 15, and 15 pg/ml, respectively.

Detection of IL-8 mRNA
Following treatment (with or without serum), the medium was aspirated and total cellular mRNA was isolated using a commercial reagent (TRIreagent; Molecular Reagents, Cincinnati, OH). For Northern analysis the isolated RNA samples were electrophoresed, blotted, and probed with P32-labeled IL-8 cDNA ("Prime it" Kit; Stratagene, La Jolla, CA) as previously described (15). The IL-8 cDNA was a generous gift of David Denhardt (Department of Cell Biology, Rutgers University). After hybridization, the blot was washed at high stringency (0.1x SSC/0.1% SDS at 50°C for 15 min), and then exposed to a phosphorimager screen for relative quantification of IL-8 and actin mRNA.

IL-8 mRNA Stability Assay
To measure IL-8 mRNA stability, BE cell monolayers were incubated with BEGM media along with serum (5 or 10%) or medium alone for 2 and 4 h. Following the incubation period, the media was aspirated and replaced with BEGM media containing 20 µg/ml daunorubicin (5,6-dichloro-1-[ß]-D-ribofuranosyl benzimidazole). After allowing 15 min for transcriptional activity to cease, cells were lysed at time 0, 15, 30, 60, 120, and 240 min thereafter, and subjected to Northern analysis as described above.

Nuclear Factor-{kappa}B Electrophoretic Mobility Shift Assay
Human BE cells were grown to 80% confluence in BEGM medium in 6-well tissue culture plates. After incubation in the presence or absence of serum, cells were washed twice with 3 ml phosphate-buffered saline and then scraped along with 1 ml phosphate-buffered saline into a microcentrifuge tube. The cells were centrifuged (1,500 rpm, 4°C, 10 min), drained, and then flash frozen in liquid nitrogen and stored at -80°C. After thawing cells on ice, 15 µl extract buffer was added to the cell pellet. The extract buffer consisted of 20 mM HEPES (pH 7.9), 350 mM NaCl, 20% glycerol, 1% IGEPAL CA-630, 1 mM MgCl2, 0.5 mM EDTA, and 0.1 mM EGTA, to which the following were added just before use: 0.5 mM dithiothreitol, 15 µg/ml aprotinin, and 1:100 protease inhibitor cocktail (P-8340; Sigma). The cells were incubated on ice for 30 min with gentle mixing every 10 min. The extract was centrifuged (14,000 rpm, 10–15 min) to remove cell particulates, and protein was measured by Bradford assay.

Nuclear factor (NF)-{kappa}B–binding reaction was performed in 15 mM Tris pH 7.5, 75 mM NaCl, 1.5 mM EDTA, 1.5 mM dithiothreitol, 7.5% glycerol, 0.3% IGEPAL CA-630, and 20 µg/ml bovine serum albumin with freshly added 50 µg/ml poly IC. After incubation (20 min) on ice an end-labeled, double-stranded oligo (5'-CTCAACAGAGGGGACTTTCCGAGAGGCAT-3') with a specific NF-{kappa}B–binding site was added and the reaction mixture was incubated (25°C) for an additional 20 min. The complexes were separated on a 4% native polyacrylamide gel. The gel was dried and exposed to X-ray film.

Data Analysis
All statistical analyses were performed with the help of computer software (SigmaPlot 2,000 and SigmaStat 2.03; SPSS, Inc., Chicago, IL). For normally-distributed data sets, repeated measures ANOVA tests were performed. Multiple comparisons versus control values were performed using Dunn's Method, or Dunnett's Method for repeated measures ANOVA. For data sets that were not normally distributed, between-group analyses were performed using ANOVA on ranks. P values of < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Serum on Epithelial Cell IL-8 Secretion
Monolayers of BE cells were incubated for 0–48 h with 0–10% human serum in BEGM, and supernatant fluids were sampled for IL-8 and G-CSF. Incubation of BE with serum promoted IL-8 secretion beginning 4 h after the serum was added, and a dose-related augmentation of IL-8 secretion continued for the entire 48 h (Figure 1). BE cells incubated with higher concentrations of serum also tended to secrete G-CSF (257 ± 141 pg/ml, 5% serum at 48 h), although this did not occur in all cell cultures and was not statistically significant. In contrast to effects on cytokines that promote neutrophil synthesis and chemotaxis, serum did not stimulate secretion of eotaxin (data not shown), suggesting that it is not a nonspecific activator of epithelial cell cytokine synthesis.



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  		Figure 1. Effect of serum on IL-8 secretion by BE cells. Monolayers of BE cells were incubated in medium (open bars) with either 1% (striped bars), 5% (hatched bars), or 10% (solid bars) serum. Supernatant fluids were sampled 0–48 h after the addition of serum to the medium and were analyzed for IL-8. The graphs represent the mean values of two separate experiments.

 
To determine the approximate molecular weight of the IL-8–inducing factor, serum was centrifuged through filters with different molecular weight cutoffs, and the filtrate was then incubated (24 h, 37°C) with BE cells. Significant IL-8–inducing activity was able to pass through filters with MW cutoffs of 100 and 500 kD, whereas virtually no activity passed through filters with a pore size of 50 kD or smaller (Figure 2). Heat treatment of the serum (56°C for 30 min) did not affect serum IL-8–inducing activity (data not shown).



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  		Figure 2. IL-8–inducing activity of serum filtrates. Human serum was centrifuged through filters with different molecular weight cutoffs (3–500 kD). Next, BE cell monolayers were incubated with either serum (0.5–5%), serum filtrates (0.5–5%), or medium alone, and supernatant fluids obtained 24 h later were analyzed for IL-8. The graphs represent the mean values ± SEM of five separate experiments (*P < 0.05 versus control samples from cells incubated in BEGM alone). Open bars, medium alone; striped bars, 0.5% serum; hatched bars, 1% serum; solid bars, 5% serum.

 
Effect of Platelet Activation and Clotting on BE IL-8–Inducing Activity
To determine whether the IL-8–inducing activity of serum was related to either platelet activation or an activated clotting factor, BE cells were incubated (24 h, 37°C) with either serum clotted in the presence of platelets, serum clotted by the addition of CaCl2 after platelets had been removed, platelet-free plasma, or medium alone, and IL-8 levels in culture supernatant fluids were determined. Serum and platelet-free serum induced the highest levels of IL-8 secretion (Figure 3). Platelet-free plasma was also a potent inducer of IL-8, although levels were ~ 60% of the levels achieved with serum. Cells incubated with either CaCl2 or medium alone secreted relatively little IL-8.



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  		Figure 3. Contribution of clotting and platelet activation to IL-8–inducing activity of serum and plasma. Monolayers of BE cells were incubated (24 h, 37°C) with either plasma that had been centrifuged to remove platelets, serum that had been clotted by the addition of CaCl2 in the absence of platelets, or serum that was clotted in the presence of platelets. Cells incubated in serum-free medium with CaCl2 served as controls. Cell supernatant fluids were then analyzed for the presence of IL-8. The graphs represent the mean values ± SEM of three separate experiments (*P < 0.05 versus medium control). Open bars, medium alone; striped bars, 0.5% serum; hatched bars, 1% serum; solid bars, 5% serum.

 
Effect of Individual Serum Proteins on IL-8 Secretion
The activity of serum on IL-8 secretion was compared with that of some of the major protein components of serum, including albumin, {gamma}-globulin, and LDL. Both serum and LDL significantly increased IL-8 secretion in a dose-related fashion (Figure 4). In contrast, neither IgG nor albumin affected IL-8 secretion at concentrations as high as 1 mg/ml. The IL-8 content of the undiluted serum and LDL preparations was < 6 pg/ml (data not shown).



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  		Figure 4. Effect of specific serum components on BE cell IL-8 secretion. Monolayers of BE cells were incubated (48 h) in medium alone, or in medium together with either serum (0.1–5%), LDL (10–500 µg/ml), human serum albumin (50–1,000 µg/ml), or IgG (50–1,000 µg/ml), and IL-8 levels were measured in supernatant fluids. The graphs represent mean values ± SEM for three separate experiments.

 
To further define the effects of LDL on IL-8 secretion, BE cells were incubated with LDL in the presence or absence of a blocking antibody specific for the LDL receptor (C7, 50 µg/ml). LDL produced a dose-related increase in IL-8 that was partially blocked (62–67% reduction) by C7 mAb (Figure 5A). This experiment was repeated using 0.5% serum or plasma to determine the contribution of LDL to the total IL-8–inducing activity. Coincubation with C7 partially inhibited the IL-8–inducing activity of 0.5% serum and plasma by 53 and 57%, respectively (Figures 5B and 5C).



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  		Figure 5. Effects of LDL receptor blockade on IL-8–inducing activity. Nearly confluent monolayers of cells were preincubated (1 h) with either C7 mAb (50 µg/ml; solid bars), an equivalent amount of an IgG2b isotype control antibody (striped bars), or medium alone (open bars). Next, the monolayers were incubated (48 h) with either (A) LDL (0–100 µg/ml), (B) 0.5% serum, or (C) 0.5% platelet-free plasma. Supernatants were collected and assayed for IL-8. The bars represent mean values ± SEM for three experiments. The data represent mean values ± SEM (n = 3 for isotype control, n = 6 for other data points, *P < 0.05 compared with samples incubated with isotype control or medium alone).

 
To determine whether a lipid in the preparation contributed to the IL-8–inducing activity of the serum, BE cells were incubated with either 0–10% serum or equivalent amounts of delipidated serum. Delipidation by either butanol/ether extraction, or by incubation with a lipid-binding resin (Aerosil), did not affect IL-8–inducing activity of serum (data not shown).

Effect of Serum on NF-{kappa}B Activation
IL-8 synthesis in airway epithelial cells can be regulated by NF-{kappa}B–dependent increases in gene transcription. To determine effects of serum on NF-{kappa}B activation, BE cells were incubated (37°C, 0–120 min) with either 5% serum, tumor necrosis factor (TNF)-{alpha} (1,000 U/ml), or medium alone, and cell extracts were prepared. Both serum and TNF-{alpha} caused rapid activation of NF-{kappa}B–binding activity that began within 15 min and peaked 60 min after the addition of the stimuli (Figure 6A). In addition, both the serum and TNF-{alpha}–induced NF-{kappa}B–binding activity were abrogated by incubation with an excess of unlabeled NF-{kappa}B–specific oligonucleotide, but not by an excess of an irrelevant oligonucleotide (Figure 6B).



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  		Figure 6. Serum activation of NF-{kappa}B. (A) Primary BE cell monolayers were incubated with 5% serum, medium alone, or TNF-{alpha} (1,000 U/ml); and cell lysates were analyzed by EMSA at 0, 15, 30, 60, and 120 min to detect activated NF-{kappa}B. The blot shown is representative of three separate experiments. (B) Extracts from cells incubated with either medium alone, 5% serum (lanes 1–3), or TNF-{alpha} (lanes 4–6) were incubated with medium alone (lanes 1 and 4), an excess of unlabeled NF-{kappa}B oligonucleotide (lanes 2 and 5), or an excess of an irrelevant oligonucleotide (containing an AP-1–binding site, lanes 3 and 6). These samples were then analyzed for NF-{kappa}B–binding activity by electrophoretic mobility shift assay.

 
Effect of Serum on IL-8 mRNA and mRNA Stability
To determine whether serum effects on IL-8 secretion were accompanied by increased IL-8 mRNA, monolayers of BE cells were incubated either in medium alone or in medium with 5–10% human serum, and cell extracts at baseline, 2, and 4 h afterwards were analyzed for IL-8 and actin mRNA. Incubation with serum for 2 or 4 h increased IL-8 mRNA compared with cells incubated in medium alone, whereas expression of actin was relatively unchanged (Figure 7A). After normalization for expression of actin mRNA, incubation with 5% serum for 4 h increased IL-8 mRNA by > 4-fold (Figure 7B).



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  		Figure 7. Effects of serum on IL-8 mRNA. Monolayers of BE cells were incubated (2 or 4 h) with serum (5 or 10%) or medium alone, and (A) IL-8 and actin mRNA were determined by Northern blot. (B) The kinetics of IL-8 mRNA were determined by analyzing the Northern blot results by densitometry, and the data are normalized to expression of actin mRNA under the same conditions (mean ± SE, n = 3, *P = 0.05 versus baseline IL-8 mRNA).

 
Next, BE cells were incubated (0–4 h, 37°C) in the presence or absence of 5% serum, and effects on IL-8 mRNA stability were determined. The t1/2 of IL-8 mRNA in BE cells incubated in serum was > 4 h (Figure 8), compared with a t1/2 of 55–60 min in cells incubated in serum-free medium.



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  		Figure 8. Effect of serum on IL-8 mRNA stability. Monolayers of BE cells were incubated with 5% serum for 2 or 4 h, or were incubated in the absence of serum. (A) Total RNA extracts were then analyzed at intervals after the addition of daunorubicin: < 2 min (lanes 2, 8, and 14), 15 min (lanes 3, 9, and 15), 30 min (lanes 4, 10, and 16), 60 min (lanes 5, 11, and 17), 120 min (lanes 6, 12, and 18), and 240 min (lanes 7 and 13). Lane 1 represents control cells that were incubated in medium alone without either serum or daunorubicin. (B) The data were quantitated by densitometry, and plotted relative to the amount of IL-8 mRNA that was detected at the time that the daunorubicin was added.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is generally assumed that exudation of serum proteins into the airway is a nonspecific response to viral infections and other inflammatory stimuli. In these experiments, serum was demonstrated to be a potent stimulus for IL-8 mRNA and secretion in BE cells. This effect does not constitute a nonspecific activation of epithelial cells, because specific serum components (LDL but not albumin or IgG) were found to upregulate IL-8 but not eotaxin. These findings suggest that specific serum proteins could provide an important innate stimulus for rapid neutrophilic responses to infections and irritants that cause increased vascular permeability in respiratory airway epithelium. This effect may be of particular importance during infections with respiratory viruses (e.g., rhinovirus), because IL-8 is an important chemoattractant and activator of neutrophils in this context (16, 17). Furthermore, neutrophilic inflammation is closely associated with respiratory symptoms, and in subjects with asthma, with virus-induced changes in lower airway physiology (18).

LDL was identified as one serum protein that induces IL-8 from BE cells, and the specificity of this effect was demonstrated by inhibition with C7 mAb, which blocks binding to the LDL receptor. The blocking antibody had less of an effect, however, on the IL-8–inducing activity of whole serum or plasma, indicating that there are other components in addition to LDL (such as products of platelet activation) that contribute to this activity. Published data indicate that LDL can also induce endothelial cells to secrete IL-8, although oxidation of LDL was required for this to occur (19). In our epithelial cell system, oxidized LDL was toxic to the cells, as has been described for other epithelial cells (20), and did not upregulate IL-8 (data not shown). These findings suggest that responses to LDL and oxidized LDL may be distinct in different types of cells.

Serum caused increases in epithelial cell IL-8 mRNA that paralleled the increase in IL-8 secretion. Because the steady state level of IL-8 mRNA can be regulated either by transcription or by changing the stability of the mRNA, we evaluated the effects of serum on IL-8 mRNA half-life, and on activation of NF-{kappa}B, which is known to be a positive regulator of IL-8 gene promoter activity (21, 22). Serum incubation rapidly prolonged mRNA stability from 55–60 min to > 4 h, and also increased NF-{kappa}B–binding activity in cell extracts. The relatively short IL-8 mRNA t1/2 in unstimulated cells is in agreement with previous studies conducted in HeLa cells, in which the half-life of IL-8 mRNA was estimated to be ~ 20 min (21, 23). Together, these data suggest that serum, which is a potent stimulus for IL-8 production, enhances both IL-8 transcription and mRNA stability.

Although these observations were made in cultured airway epithelial cells, there is evidence from clinical studies that serum proteins can attain concentrations in the airways that are similar to those used in these experiments. Serum proteins such as albumin, IgG, ceruloplasmin, and {alpha}2 macroglobulin have been measured in airway fluids obtained by nasal lavage, sputum induction, and bronchoalveolar lavage, and can be increased by infections, exposure to allergens or irritants (e.g., tobacco smoke, ozone, histamine), and by the neurokinin 1 (NK1) receptor agonist substance P (24–29). For example, levels of albumin in nasal lavage fluid are low in healthy individuals, but can increase 10- to 15-fold during an acute viral infection, and attain an average concentration of 200–500 µg/ml during the peak of the cold (7, 30, 31). Because normal serum albumin levels are 3.5–5.0 g/l (3,500–5,000 µg/ml), albumin concentrations in nasal lavage fluid obtained during respiratory infections are ~ 5–10% of typical serum albumin levels. Considering the dilution that occurs during nasal lavage, the actual levels of albumin in nasal secretions are even higher, suggesting that the concentrations of serum proteins used in our in vitro experiments are representative of airway physiology during acute viral infections. Concentrations of albumin in sputum are also elevated during viral infections (31, 32) and in asthma (33, 34). Interestingly, the concentrations of albumin in asthma correlate with asthma severity (33) and asthma symptoms (34), and are inversely related to measures of airway obstruction (FEV1/FVC ratio) (33). Whether LDL is also present in high levels in airway secretions has not yet been determined.

Interestingly, one of the cellular receptors for LDL is also used by minor group rhinoviruses to gain entry to host epithelial cells (35). Accordingly, experiments using cultured epithelial cells have shown that LDL can inhibit cytopathic effect caused by minor group rhinoviruses, but not major group viruses that instead bind to intracellular adhesion molecule-1 (13). This suggests the possibility that during infections with minor group rhinoviruses, transudation of LDL into the airway could serve to inhibit the cell-to-cell spread of the infection.

Viral infections have been shown to induce IL-8 through several different pathways including binding to surface receptors, replication, and changes in oxidation state (13, 36, 37). In addition, double-stranded RNA, which is likely involved in triggering intracellular innate responses during viral replication, also binds to cell membranes via toll-like receptor-3 (TLR-3) (38), and is a potent inducer of IL-8 (39). When compared with these other mechanisms, our data suggest that the effect of serum is likely to be a quantitatively important contributor to upregulation of IL-8 during viral infections. Finally, the multiple and redundant pathways for IL-8 induction suggest that, in addition to being a stimulus for inflammation, this chemokine is an important component of the host antiviral response.

In summary, we have found that serum and LDL are potent inducers of IL-8 production in nontransformed airway epithelial cells. Because a wide variety of inflammatory disorders are associated with leakage of serum proteins into airway secretions, this effect could represent an innate mechanism for the recruitment of neutrophils to areas of inflammation in response to infections, allergens, or irritants. In many situations, this may be a protective effect that is beneficial to the host; however, an overexuberant neutrophilic response could add to airway pathology and subsequent morbidity. Identification of the major factors that are responsible for this effect could lead to new therapeutic responses for a variety of airway inflammatory conditions.


   Acknowledgments
 
This work was supported by NIH grants HL/AI60993, P01 HL70831, and P01 AI35891.

Received in original form December 18, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mosser, A. G., R. A. Brockman-Schneider, S. P. Amineva, L. Burchell, J. B. Sedgwick, W. W. Busse, and J. E. Gern. 2002. Similar frequency of rhinovirus-infectable cells in upper and lower airway epithelium. J. Infect. Dis. 185:734–743.[CrossRef][Medline]
  2. Arruda, E., T. R. Boyle, B. Winther, D. C. Pevear, J. M. Gwaltney, and F. G. Hayden. 1995. Localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization. J. Infect. Dis. 171:1329–1333.[Medline]
  3. Hendley, J. O. 1998. The host response, not the virus, causes the symptoms of the common cold. Clin. Infect. Dis. 26:847–848.[Medline]
  4. Johnston, S. L., P. K. Pattemore, G. Sanderson, S. Smith, F. Lampe, L. Josephs, P. Symington, S. O'Toole, S. H. Myint, D. A. Tyrrell, and S. T. Holgate. 1995. Community study of role of viral infections in exacerbations of asthma in 9–11 year old children. BMJ 310:1225–1229.[Abstract/Free Full Text]
  5. Seemungal, T. A., R. Harper-Owen, A. Bhowmik, D. J. Jeffries, and J. A. Wedzicha. 2000. Detection of rhinovirus in induced sputum at exacerbation of chronic obstructive pulmonary disease. Eur. Respir. J. 16:677–683.[Abstract]
  6. Smyth, A. R., R. L. Smyth, C. Y. W. Tong, C. A. Hart, and D. P. Heaf. 1995. Effect of respiratory virus infections including rhinovirus on clinical status in cystic fibrosis. Arch. Dis. Child. 73:117–120.[Abstract]
  7. Igarashi, Y., D. P. Skoner, W. J. Doyle, M. V. White, P. Fireman, and M. A. Kaliner. 1993. Analysis of nasal secretions during experimental rhinovirus upper respiratory infections. J. Allergy Clin. Immunol. 92:722–731.[CrossRef][Medline]
  8. Cheung, D., E. C. Dick, M. C. Timmers, E. P. A. De Klerk, W. J. M. Spaan, and P. J. Sterk. 1995. Rhinovirus inhalation causes long-lasting excessive airway narrowing in response to methacholine in asthmatic subjects in vivo. Am. J. Respir. Crit. Care Med. 152:1490–1496.[Abstract]
  9. Yuta, A., W. J. Doyle, E. Gaumond, M. Ali, L. Tamarkin, J. N. Baraniuk, M. Van Deusen, S. Cohen, and D. P. Skoner. 1998. Rhinovirus infection induces mucus hypersecretion. Am. J. Physiol. Lung Cell. Mol. Physiol. 274:L1017–L1023.[Abstract/Free Full Text]
  10. Levandowski, R. A., C. W. Weaver, and G. G. Jackson. 1988. Nasal secretion leukocyte populations determined by flow cytometry during acute rhinovirus infection. J. Med. Virol. 25:423–432.[Medline]
  11. Gern, J. E., and W. W. Busse. 2000. The role of viral infections in the natural history of asthma. J. Allergy Clin. Immunol. 106:201–212.[Medline]
  12. Turner, R. B., K. W. Weingand, C. H. Yeh, and D. W. Leedy. 1998. Association between interleukin-8 concentration in nasal secretions and severity of symptoms of experimental rhinovirus colds. Clin. Infect. Dis. 26:840–846.[Medline]
  13. Schroth, M. K., E. Grimm, P. Frindt, D. M. Galagan, S. Konno, R. Love, and J. E. Gern. 1999. Rhinovirus replication causes RANTES production in primary bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 20:1220–1228.[Abstract/Free Full Text]
  14. Cham, B. E., and B. R. Knowles. 1976. A solvent system for delipidation of plasma or serum without protein precipitation. J. Lipid Res. 17:176–181.[Abstract]
  15. Bhattacharya, S., T. Giordano, G. Brewer, and J. S. Malter. 1999. Identification of AUF-1 ligands reveals vast diversity of early response gene mRNAs. Nucleic Acids Res. 27:1464–1472.[Abstract/Free Full Text]
  16. Teran, L. M., S. L. Johnston, J. M. Schroder, M. K. Church, and S. T. Holgate. 1997. Role of nasal interleukin-8 in neutrophil recruitment and activation in children with virus-induced asthma. Am. J. Respir. Crit. Care Med. 155:1362–1366.[Abstract]
  17. Gern, J. E., R. Vrtis, K. A. Grindle, C. Swenson, and W. W. Busse. 2000. Relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection. Am. J. Respir. Crit. Care Med. 162:2226–2231.[Abstract/Free Full Text]
  18. Gern, J. E., and W. W. Busse. 2002. Relationship of viral infections to wheezing illnesses and asthma. Nat. Rev. Immunol. 2:132–138.[CrossRef][Medline]
  19. Claise, C., M. Edeas, J. Chalas, A. Cockx, A. Abella, L. Capel, and A. Lindenbaum. 1996. Oxidized low-density lipoprotein induces the production of interleukin-8 by endothelial cells. FEBS Lett. 398:223–227.[CrossRef][Medline]
  20. Masella, R., E. Straface, C. Giovannini, R. Di Benedetto, B. Scazzocchio, M. Viora, A. Cantafora, and W. Malorni. 2000. Subcellular alterations induced by UV-oxidized low-density lipoproteins in epithelial cells can be counteracted by alpha-tocopherol. Photochem. Photobiol. 71:97–102.[CrossRef][Medline]
  21. Holtmann, H., R. Winzen, P. Holland, S. Eickemeier, E. Hoffmann, D. Wallach, N. L. Malinin, J. A. Cooper, K. Resch, and M. Kracht. 1999. Induction of interleukin-8 synthesis integrates effects on transcription and mRNA degradation from at least three different cyto. Mol. Cell. Biol. 19:6742–6753.[Abstract/Free Full Text]
  22. Roebuck, K. A. 1999. Regulation of interleukin-8 gene expression. J. Interferon Cytokine Res. 19:429–438.[CrossRef][Medline]
  23. Winzen, R., M. Kracht, B. Ritter, A. Wilhelm, C. Y. Chen, A. B. Shyu, M. Muller, M. Gaestel, K. Resch, and H. Holtmann. 1999. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J. 18:4969–4980.[CrossRef][Medline]
  24. Pizzichini, M. M., E. Pizzichini, A. Efthimiadis, L. Clelland, J. B. Mahony, J. Dolovich, and F. E. Hargreave. 1997. Markers of inflammation in induced sputum in acute bronchitis caused by Chlamydia pneumoniae. Thorax 52:929–931.[Abstract]
  25. Gorski, P., A. Krakowiak, and U. Ruta. 2000. Nasal and bronchial responses to flour-inhalation in subjects with occupationally induced allergy affecting the airway. Int. Arch. Occup. Environ. Health 73:488–497.[CrossRef][Medline]
  26. Hill, A. T., E. J. Campbell, S. L. Hill, D. L. Bayley, and R. A. Stockley. 2000. Association between airway bacterial load and markers of airway inflammation in patients with stable chronic bronchitis. Am. J. Med. 109:288–295.[CrossRef][Medline]
  27. Newson, E. J., M. T. Krishna, L. C. Lau, P. H. Howarth, S. T. Holgate, and A. J. Frew. 2000. Effects of short-term exposure to 0.2 ppm ozone on biomarkers of inflammation in sputum, exhaled nitric oxide, and lung function in subjects with mild atopic asthma. J. Occup. Environ. Med. 42:270–277.[Medline]
  28. Braun, J., A. Mehnert, K. Dalhoff, K. J. Wiessmann, and F. Ratjen. 1997. Different BALF protein composition in normal children and adults. Respiration (Herrlisheim) 64:350–357.
  29. Van Rensen, E. L., P. S. Hiemstra, K. F. Rabe, and P. J. Sterk. 2002. Assessment of microvascular leakage via sputum induction: the role of substance P and neurokinin A in patients with asthma. Am. J. Respir. Crit. Care Med. 165:1275–1279.[Abstract/Free Full Text]
  30. Proud, D., J. M. Jr. Gwaltney, J. O. Hendley, C. A. Dinarello, S. Gillis, and R. P. Schleimer. 1994. Increased levels of interleukin-1 are detected in nasal secretions of volunteers during experimental rhinovirus colds. J. Infect. Dis. 169:1007–1013.[Medline]
  31. Fahy, J. V., K. W. Kim, J. Liu, and H. A. Boushey. 1995. Prominent neutrophilic inflammation in sputum from subjects with asthma exacerbation. J. Allergy Clin. Immunol. 95:843–852.[CrossRef][Medline]
  32. Pizzichini, M. M., E. Pizzichini, A. Efthimiadis, A. J. Chauhan, S. L. Johnston, P. Hussack, J. Mahony, J. Dolovich, and F. E. Hargreave. 1998. Asthma and natural colds. Inflammatory indices in induced sputum: a feasibility study. Am. J. Respir. Crit. Care Med. 158:1178–1184.[Abstract/Free Full Text]
  33. Louis, R., L. C. Lau, A. O. Bron, A. C. Roldaan, M. Radermecker, and R. Djukanovic. 2000. The relationship between airways inflammation and asthma severity. Am. J. Respir. Crit. Care Med. 161:9–16.[Abstract/Free Full Text]
  34. Pizzichini, E., M. M. Pizzichini, A. Efthimiadis, S. Evans, M. M. Morris, D. Squillace, G. J. Gleich, J. Dolovich, and F. E. Hargreave. 1996. Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements. Am. J. Respir. Crit. Care Med. 154:308–317.[Abstract]
  35. Gruenberger, M., R. Wandl, J. Nimpf, T. Hiesberger, W. J. Schneider, E. Kuechler, and D. Blaas. 1995. Avian homologs of the mammalian low-density lipoprotein receptor family bind minor receptor group human rhinovirus. J. Virol. 69:7244–7247.[Abstract]
  36. Biagioli, M. C., P. Kaul, I. Singh, and R. B. Turner. 1999. The role of oxidative stress in rhinovirus induced elaboration of IL-8 by respiratory epithelial cells. Free Radic. Biol. Med. 26:454–462.[CrossRef][Medline]
  37. Kaul, P., I. Singh, and R. B. Turner. 1999. Effect of nitric oxide on rhinovirus replication and virus-induced interleukin-8 elaboration. Am. J. Respir. Crit. Care Med. 159:1193–1198.[Abstract/Free Full Text]
  38. Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition of double-stranded RNA and activation of NF-kappaB by Toll- like receptor 3. Nature 413:732–738.[CrossRef][Medline]
  39. Gern, J. E., D. A. French, K. A. Grindle, R. A. Brockman-Schneider, S. Konno, and W. W. Busse. 2003. Double-stranded RNA induced the synthesis of specific chemokines by bronchial epithelial cells. Am. J. Respir. Crit. Care Med. 28:731–737.



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