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Clin Exp Immunol. Jun 2006; 144(3): 534–542.
PMCID: PMC1941973

Induction of CXC chemokines in A549 airway epithelial cells by trypsin and staphylococcal proteases − a possible route for neutrophilic inflammation in chronic rhinosinusitis

Abstract

While various microorganisms have been recovered from patients with chronic rhinosinusitis, the inflammatory impact of virulence factors, in particular proteases from Staphylococcus aureus and coagulase negative staphylococci on the nasal epithelium, has not yet been investigated. Expression of CXC chemokines was determined in the epithelium of patients with chronic rhinosinusitis by immunohistochemistry. In a cell culture system of A549 respiratory epithelial cells, chemokine levels were quantified by enzyme-linked immunosorbent assay (ELISA) after stimulation with supernatants originating from three different staphylococcal strains or with trypsin, representing a serine protease. Inhibition experiments were performed with prednisolone, with the serine protease inhibitor 4-(2-aminoethyl)-benzenesulphonylfluoride (AEBSF) and with the nuclear transcription factor (NF)-κΒ inhibitor (2E)-3-[[4-(1,1-dimethylethyl)phenyl]sulphonyl]-2-propenenitrite (BAY) 11–7085. Electromobility shift assays (EMSA) were used to demonstrate NF-κB-dependent protein synthesis. CXC chemokines interleukin (IL)-8, growth-related oncogene alpha (GRO-α) and granulocyte chemotactic protein-2 (GCP-2) were expressed in the patients’ epithelium whereas epithelial cell-derived neutrophil attractant 78 (ENA-78) was rarely detected. In A549 cells, chemokines IL-8, ENA-78 and GRO-α but not GCP-2 were induced by trypsin and almost equal levels were induced by staphylococcal supernatants. IL-8, GRO-α and ENA-78 synthesis was suppressed almost completely by AEBSF and BAY 11–7085, whereas prednisolone reduced chemokine levels differentially dependent on the supernatant added. CXC chemokines were detectable in the epithelium of patients with chronic rhinosinusitis. Staphylococcal serine proteases induced CXC chemokines in A549 cells, probably by the activation of proteases activated receptors, and thus might potentially be involved in neutrophilic inflammation in chronic sinusitis.

Keywords: A549, chemokines, PAR, sinusitis, Staphylococcus aureus

Introduction

Sinusitis is an inflammatory process involving one or more sinuses, and is divided commonly into acute, recurrent and chronic forms [1]. Nasal polyposis (NP) represents a severe subform of chronic rhinosinusitis and is characterized clinically by nasal polyps originating from the middle nasal meatus and/or from the anterior ethmoid. In most cases of bilateral diffuse NP histology revealed a marked tissue infiltration with eosinophils and to a lesser degree with neutrophils and other inflammatory cells. In contrast, in tissue specimens of patients suffering from chronic rhinosinusitis without nasal polyps (CRS), more neutrophils than eosinophils were detected [2], assuming that CRS and NP are different entities of chronic rhinosinusitis. Another striking phenomenon reveals that permanent cure of patients suffering from NP, in particular, cannot be achieved, neither with sinus surgery nor with application of steroids, and the reasons are therefore unknown. As in allergic rhinitis and asthma, little is known about the role of neutrophils. Here, it has been assumed that neutrophil chemokines are regulated differently by steroids and that failure of this regulation might be one reason for persistence of lung neutrophilia.

Because more than 50% of neutrophils were activated due to neutrophil-elastase positivity [2] in NP and CRS, we focused on possible routes of regulation mechanisms of neutrophil-attracting CXC chemokines. For this purpose we took the presence of bacteria in the upper airway into account, as the impact of these microorganisms on the onset of chronic rhinosinusitis still remains unclear. Recent reports revealed that a wide spectrum of bacterial species has been recovered from patients with chronic rhinosinusitis, among them Staphylococcus aureus, coagulase-negative staphylococci (CoNS), Pseudomonas aeruginosa and anaerobic bacteria [35]. While it has been demonstrated that S. aureus enterotoxins may act as superantigens, thereby inducing a topical multi-clonal IgE-formation as well as a severe, possibly steroid-insensitive eosinophilic inflammation in NP [6], further research is warranted to study the effects of other major staphylococcal virulence factors such as proteases on the airway epithelium in chronic rhinosinusitis. In particular, soluble serine proteases are known to initiate and maintain inflammatory mechanisms by activating specific G protein-coupled receptors, defined as protease activated receptors (PARs). Activation of PARs leads to G-protein regulated gene transcription responses that in turn induce the production of cytokines and chemokines [7].

As a first step, we investigated expression profiles of CXC chemokines growth-related oncogene alpha (GRO-α/CXCL1), epithelial cell-derived neutrophil attractant 78 (ENA-78/CXCL5), granulocyte chemotactic protein-2 (GCP-2/CXCL6) and interleukin (IL)-8/CXCL8 in the nasal epithelium of patients suffering from chronic rhinosinusitis. These factors were selected because the epithelial layer of the nasal/paranasal mucosa represents the first line of defence against microorganisms, and induction of CXC chemokines has been observed in bacterial-induced airway inflammation [8].

Subsequently, in a cell culture model of A549 airway epithelial cells known to express PAR-1 to PAR-4 [9], IL-8, ENA-78, GRO-α and GCP-2 expression was induced by the PAR-2/PAR-4 agonist trypsin and quantified after 6 and 24 h. In addition, three different staphylococcal supernatants were used to investigate their impact on the expression of CXC chemokines in A549 airway epithelial cells. Finally, chemokine responses were modulated by the serine protease inhibitor 4-(2-aminoethyl)-benzenesulphonylfluoride (AEBSF) or by prednisolone and the involvement of nuclear transcription factor κΒ (NF-κΒ) on trypsin- and staphylococcal-mediated expression of neutrophil chemokines was investigated by electromobility shift assays (EMSA).

Materials and methods

Trypsin, and unless otherwise stated, all other reagents used, were purchased from Sigma (Deisenhofen, Germany).

Patients

Fifteen CRS specimens [recovered from seven males, mean age ± standard deviation (s.d.), 52·60 ± 24·93 years, eight females, 55·71 ± 18·71, all of them non-allergic, non-smokers], 15 NP specimens (eight males, 65·60 ± 16·92, seven females 57·80 ± 10·43, all of them non-allergic, non-smokers, no aspirin-sensitivity) and 15 turbinate mucosa (TM) specimens (seven males, 40·15 ± 18·72, eight females 36·86 ± 12·86, all of them non-allergic, non-smokers) were embedded in paraffin.

Chronic rhinosinusitis was diagnosed according to the medical history with corresponding clinical symptoms, a preoperative computerized tomography (CT) scan and an endoscopic examination of the nasal cavity. Those cases in which nasal polyps were present in the middle nasal meatus were classified as NP. TM was obtained from patients undergoing septoplasty with no history of rhinosinusitis. No patient had undergone sinus surgery previously. Allergy was excluded by negative skin-prick tests to common inhalant allergens and radio allergy sorbent test (RAST). No patient had been treated with systemic/topical steroids or antibiotics 4 weeks prior to surgery. Informed consent was obtained from all patients and the study was approved by the ethics committee of the University of Muenster, Germany.

Immunohistochemistry

Paraffin was resolved from sections using decreasing concentrations of ethanol. Subsequent steps were performed according to the instructions of the Dako-LSAB kit (Dako-LSAB + system HRP kit (Universal), no. K0679; Dako Hamburg, Germany) as described previously for chemokines. Briefly, after inhibition of the endogenous peroxidase, polyclonal antibodies were added (IL-8: polyclonal rabbit–human IL-8 antibody, 1 : 50, AHC0881, Biosource, Solingen, Germany; ENA-78: polyclonal goat–human ENA-78 antibody, 1 : 200, AF254, R&D, Wiesbaden, Germany; GRO-α: polyclonal goat–human antibody GRO-α, 1 : 50, SC-1374, Santa Cruz, Heidelberg, Germany; GCP-2: polyclonal goat–human GCP-2 antibody, 1 : 100, SC-5813, Santa Cruz). These primary antibodies were replaced by a non-immune serum of the first antibodies for the negative control.

A universal biotinylated link-antibody served as secondary antibody. Subsequently, streptavidin peroxidase (streptavidin conjugated to horseradish peroxidase, HRPOD, Dako) was added. Sections were then incubated with diaminobenzidine (DAB) chromogen (Dako) substrate for 5 min. With regard to GRO-α and GCP-2, AEC chromogen substrate (Dako) was added for 30 min. Finally, sections were washed with Mayer’s haematoxylin (Merck, Darmstadt, Germany) for 20 s.

Microscopic quantification of stained cells

Positive-stained cells were investigated analysing the surface epithelium of TM (n = 15), CRS (n = 15) and NP (n = 15) by two independent investigators. Stained cells were counted in four randomly observed microscopic fields at 1000× magnification and divided by the total number of epithelial cells in that field. The mean of the four ratios was the count for each sample. Data were analysed using the non-parametric Mann–Whitney U-test and shown as means ± s.d. The level of significance was set at P < 0·05 (*).

Cultivation of A549 cells

A549 cells (ATTC CCL-185) were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in Hams F12K medium (gibco Life Technologies, Eggenstein, Germany) and supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma). A549 cells were cultured at 37°C, gassed with 5% CO2 and grown to 80% confluence. Finally, they were split using trypsin (0·1%).

Bacterial strains

For stimulation experiments, the well-characterized S. aureus laboratory strains Newman and COL and the reference S. epidermidis strain DSM 20044 were used. S. aureus COL, a methicillin-resistant strain (mecA gene-positive), was tested polymerase chain reaction (PCR)-positive for enterotoxin B gene (seb), whereas the Newman strain was positive for enterotoxin A gene (sea) [10]. In a previous study using an in vitro assay for protease activity in culture supernatants, S. aureus Newman produced 240 U/mg protein or 180 U/1 × 108 colony-forming units (CFU) [11]. The S. epidermidis strain used is superantigen negative [12].

Stimulation of A549 cells

Prior to stimulation, cells were incubated with Hams F12K medium devoid of FCS for 24 h. Characterization and purity of A549 cells was examined by inverted-phase contrast microscopy. Cell viability assessed by trypan blue exclusion was greater than 95% in all experiments. Staphylococcal strains were grown in 150 ml tryptic soy bouillon (TSB) for 10–12 h, adjusted to OD = 1·0 with sterile TSB, an aliquot removed for CFU determination (5 × 108−1 × 109), and the bacteria removed by centrifugation (3000 g for 20 min, 4°C). Supernatants of S. aureus COL and S. epidermidis DSM20044 were added in a dilution of 1 : 5. Supernatants of S aureus Newman D2C (ATCC 25904) were employed in a dilution of 1 : 10.

Inhibition was performed with prednisolone (Merck) at a concentration of 10 µM or with the serine protease inhibitor AEBSF [4-(2-aminoethyl)-benzenesulphonyl fluoride] at a concentration of 1 mM.

BAY 11–7085 [(2E)-3-4-1,1-dimethylethylphenylsulphonyl-2-propenenitrile], which represents an inhibitor of ΝF-κΒ, was employed at a concentration of 10 mm to block NF-κB dependent synthesis of CXC chemokines.

Protein measurements

After 6 and 24 h, supernatants were harvested and enzyme-linked immubosorbent assay (ELISA) tests for IL-8 (detection range > 3·5 pg/ml), ENA-78 (detection range > 15 pg/ml), GRO-α (detection range > 10 pg/ml) and GCP-2 (detection range > 1·6 pg/ml) were performed in duplicate according to the manufacturer’s instructions (R&D Systems, Wiesbaden, Germany). The optical density of the samples was measured at a wavelength of 450 nm. Protein levels were tested using the non-parametric Mann–Whitney U-test. Results were shown as mean ± s.d. P-values < 0·05 (*) were considered statistically significant.

EMSA

The assay was performed as described recently with minor modifications [13]. Briefly, after treatment of A549 with S. aureus Newman and trypsin for 30 and 60 min, cells were washed with phosphate-buffered saline (PBS) and lysed in 60 µl of total cell extract buffer (20 mm HEPES pH 7·9, 350 mM NaCl, 1 mM MgCl2, 0·5 mM ethylenediamine tetraacetic acid (EDTA), 0·1 mm ethylene-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 20% (v/v) glycerol, 1% (v/v) Nonidet P-40, 0·5 mM dithiothreitol (DTT), 2 mM phenylmethylsulphonyl fluoride (PMSF), 50 µg/ml aprotinin, 50 µg/ml leupeptin). The lysate-containing proteins was cleared by centrifugation and the supernatant was stored at −80°C after the protein concentration had been determined. The binding reaction was carried out in 20 µl of the mixture containing 4 µl of cell extract (10 µg protein), 4 µl of 5× binding buffer (20 mM HEPES, pH 7·7, 50 mM KCl, 1 mM DTT, 2·5 mM MgCl2 and 20% Ficoll), 2 µg poly(dI-dC) (Roche Diagnostics, Mannheim, Germany) as a non-specific competitor DNA, 2 µg of bovine serum albumin (BSA), and 80 000 counts per minute (cpm) of the labelled oligonucleotide. After 20 min of incubation at room temperature, the samples were electrophoresed through a 4% non-denaturing polyacrylamide gel. The gel was then dried and exposed to Kodak BioMax XAR film. To verify specific NF-κB activity, competitive EMSA with a 30-fold excess of unlabelled oligonucleotide was used in the binding reaction and allowed to react for 20 min before adding [32P]-end-labelled oligonucleotide.

The NF-κB-binding oligonucleotide (Santa Cruz Biotechnology) was end-labelled using [γ-32P] ATP (Hartmann Analytic, Braunschweig, Germany) by T4 polynucleotide kinase (Fermentas Life Sciences, St Leon-Rot, Germany) according to the manufacturer’s instructions. The labelled oligonucleotide was purified using the nucleotide removal kit (Qiagen, Hilden, Germany).

Results

Immunohistochemistry of nasal epithelium

IL-8-, GRO-α- and GCP-2-stained cells were increased significantly in the epithelium of CRS patients compared to TM. A similar pattern of IL-8, GRO-α and GCP-2 expression was found for NP. However, IL-8 and GCP-2 expression in NP was not significantly different from that in TM. ENA-78 expression was rarely observed and did not differ significantly between CRS, NP and TM. GCP-2 expression was found to be increased in the epithelium of CRS compared to NP (Figs 1 and and22).

Fig. 1
Epithelial expression of interleukin (IL)-8 in (a), expression of growth-related oncogene alpha (GRO-α) in (b), expression of epithelial cell-derived neutrophil attractant 78 (ENA-78) in (c) and expression of granulocyte chemotactic protein-2 ...
Fig. 2
Expression of neutrophil chemokines in the nasal epithelium. Data represent mean ratios (stained cells/total epithelial cells) of turbinate mucosa (TM, n = 15), chronic rhinosinusitis (CRS, n = 15) and nasal polyposis (NP, n = 15). Cells were counted ...

A549 cells release IL-8, GRO-α and ENA-78, but not GCP-2

Trypsin is a serine protease and activates PAR-2 and PAR-4 by cleaving the receptor. A significant increase of IL-8, ENA-78 and GRO-α production after 6 and 24 h was noted. Highest responses were determined for ENA-78 followed by IL-8 after 24 h. GRO-α production increased slightly between 6 and 24 h, but remained low compared to ENA-78 (Fig. 3).

Fig. 3
Release of CXC chemokines from A549 cells in response to trypsin. Trypsin-induced chemokine expression in A549 cells after 6 and 24 h (enzyme-linked immunosorbent assay). Bars represent means ± standard deviation of four independent experiments ...

In contrast to IL-8, GRO-α and ENA-78, only very low but yet detectable amounts of GCP-2 were released upon stimulation with trypsin. In order to stimulate the synthesis of GCP-2, IL-1β and lipopolysaccharide (LPS) were added to the cell culture. It was found that GCP-2 synthesis was weakly driven up by the classical proinflammatory cytokine IL-1β, but not by LPS stimulation (results not shown).

Staphylococcal supernatants induce IL-8, ENA-78 and GRO-α expression in A549 cells

Addition of S. aureus COL supernatants resulted in a significant increase in the production of IL-8, ENA-78 and GRO-α after 6 and 24 h. Again, the strongest chemokine responses were determined for ENA-78 after 24 h. Interestingly, addition of S. epidermidis DSM20044 and S. aureus COL supernatants caused a stronger IL-8, ENA-78 and GRO-α response after 6 and 24 h than addition of S. aureus Newman supernatants (Fig. 4).

Fig. 4
Induction of interleukin (IL)-8, epithelial cell-derived neutrophil attractant 78 (ENA-78) and growth-related oncogene alpha (GRO-α) by stapylococcal supernatants. Induction of neutrophil chemokines by staphylococcal supernatants in A549 cells ...

Chemokine production is inhibited by prednisolone, AEBSF and BAY 11–7085

Production of IL-8, ENA-78 and GRO-α was reduced significantly by prednisolone, AEBSF and BAY 11–7085 in all experiments (Fig. 5). Prednisolone displayed different efficacy in the inhibition of chemokines induced by staphylococcal supernatants.

Fig. 5
Inhibition of interleukin (IL)-8, epithelial cell-derived neutrophil attractant 78 (ENA-78) and growth-related oncogene alpha (GRO-α) synthesis in A549 cells by prednisolone, 4-(2-aminoethyl)-benzenesulphonylfluoride (AEBSF) and BAY 11–7085. ...

Addition of AEBSF caused almost total inhibition of IL-8, ENA-78 and GRO-α responses induced by S. epidermidis DSM20044 and S. aureus Newman, whereas S. aureus COL stimulation was inhibited by 50–60% only. Remarkably, induction of IL-8, ENA-78 and GRO-α by staphylococcal supernatants was inhibited more effectively by AEBSF than by prednisolone, thus implying protease-mediated chemokine synthesis (Fig. 5). Addition of BAY 11–7085, an inhibitor of NF-κB, caused almost complete blockage of chemokine synthesis independent of the applied staphylococcal supernatant.

Toxic effects of the applied inductors and inhibitors were considered observing the morphology of A549 cells after 24 h of stimulation. Shrinkage of cells was always visible. Desquamation of cells was pronounced if cells were stimulated with S. aureus COL or S. aureus Newman. Cell death was not observed in our experiments, with the exception of a low rate of 3% in the case of AEBSF.

Trypsin and S. aureus Newman signalling in human nasal mucosa cells via transcription factor NF-κB

EMSAs were performed with a [32P]-end-labelled oligonucleotide containing the NF-κB binding site. The PAR-2 and PAR-4 agonist trypsin (lane 4) showed a protein DNA complex band at the same level as the positive control [tumour necrosis factor (TNF)-α, lane 1]. Supernatants of S. aureus Newman showed a similar protein complex to TNF-α and trypsin. AEBSF caused an intensity loss of the band (lanes 5 and 7), indicating a reduction of the protein DNA complex. Pretreatment of the cells with prednisolone caused a reduced intensity of the band (lanes 3 and 8) (Fig. 6).

Fig. 6
Activation of nuclear transcription factor (NF)-κB in A549 cells. Lane 1: tumour necrosis factor (TNF)-α (positive control), lane 2: negative control. Lanes 3–5: stimulation with trypsin and inhibition with prednisolone (+ prds) ...

Our data reveal that specific PAR-2 and PAR-4 stimulus, trypsin and supernatants of S. aureus Newman induced activation of NF-κB in A549 cells.

Discussion

The aetiology of chronic rhinosinusitis is still poorly understood. Most recently, findings point at bacterial or microbial products that may be involved in the initiation or maintenance of chronic rhinosinusitis by the modulation of proinflammatory mediators and the initiation of tissue remodelling [1416]. Paranasal sinuses are believed to be sterile, although the upper respiratory tract, anterior nares and nasopharynx are colonized with numerous microorganisms forming the transient and resident microflora, among them staphylococci, streptococci and Corynebacterium species [17]. While CoNS are the most commonly reported microorganisms in patients with chronic rhinosinusitis, their significance is still a matter of debate [3,4,1823].

Generally, the virulence of S. aureus and in part of CoNS is determined by cell wall proteins as well as secreted toxins and enzymes. Concerning S. epidermidis, an extracellular metalloprotease with elastase activity has been detected [24]. Furthermore, an extracellular serine protease is involved in the processing of epidermin, structurally an antibiotic peptide known to be important for colonization mechanisms on mucosal surfaces [23].

With regard to S. aureus, extra-bacterial proteins that contribute to its virulence comprise adhesion proteins such as the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), a broad range of exotoxins (e.g. superantigens, exfoliative toxins and pore-forming toxins) and enzymes (e.g. proteases, lipases and nucleases) [25,26]. Gene expression of virulence factors is very complex and regulated mainly at the transcriptional level. Factors that influence gene expression are regulated largely by two-component regulatory systems such as the agr (accessory gene regulator) system. Examples of agr-regulated genes include S. aureus haemolysins (hla, hlb, hld, hlg), TSST-1 (tst) and enterotoxins B-C [27,28]. Moreover, gene expression of several proteases (slp A, B, C, D, E, F genes) and of the serine protease SspA has been shown to be agr-regulated [29,30]. Dunman et al. have identified more than 100 genes that are up-regulated and 34 that are down-regulated by the agr-system, reflecting the enormous complexity of virulence determinants in S. aureus[31]. Although it has been demonstrated that S. aureus enterotoxins may contribute to the pathogenesis of nasal polyposis [6,32,33], little is known about the role of other staphylococcal virulence factors such as serine proteases.

We therefore investigated the impact of staphylococcal proteases on the induction of CXC chemokines in A549 airway epithelial cells as epithelial cells are not considered merely as a barrier against microorganisms and environmental influences, but are also capable to modulate inflammatory responses actively by the generation of cytokines and chemokines which, in turn, attract leucocytes at sites of inflammation [34]. Previous studies have revealed that the epithelium of the respiratory reacts against luminal antigens and environmental factors by the production of proinflammatory signals as, for instance, IL-8 synthesis [35,36].

In the present study we could demonstrate that the PAR-2 agonist trypsin significantly increased IL-8, ENA-78 and GRO-α responses in A549 airway epithelial cells after 6 and 24 h of stimulation, as demonstrated previously by others [9,37]. Stimulation of A549 cells with trypsin was associated with the activation of NF-κB as shown by EMSA, and chemokine expression was completely inhibited by the NF-κB inhibitor BAY 11–7085. These results indicated that trypsin-induced chemokine expression was dependent on NF-κB, as demonstrated by Page and coworkers [38].

To investigate the impact of staphylococcal proteases on chemokine release, we performed inhibition experiments using the serine protease inhibitor AEBSF. Chemokine expression could almost be suppressed by AEBSF, more effective in experiments applying S. aureus Newman and in S. epidermidis DSM20044 than in those using S. aureus COL, thus indicating the importance of bacterial serine proteases as stimulators of chemokine synthesis in our cell culture model. Protease activation has been considered important for airway inflammation and airway remodelling. Among the four different PARs identified so far, PAR-2 has been exceedingly characterized in recent years and was found to be increased in the epithelial cells of patients with asthma [39]. Expression of CXC chemokines as a result of activation of PARs by staphylococcal proteases might therefore represent a possible mechanism to explain neutrophilic airway inflammation in chronic rhinosinusitis.

Prednisolone was tested for its ability to abolish chemokine synthesis, as topical corticosteroids are recommended for the treatment of acute and chronic rhinosinusitis [1,15]. We have shown previously that treatment of nasal polyps with prednisolone resulted in a down-regulation of IL-5 [40]. Even production of inflammatory mediators and recruitment of leucocytes in response to a Gram-negative bacterial toxin and staphylococcal enterotoxin have been shown to be sensitive to glucocorticoid treatment [41]. In this study, addition of prednisolone caused a reduction but not a complete suppression of chemokine production, in particular using S. aureus COL and S. epidermidis as bacterial strains. Very recently, it has been demonstrated that IL-8 expression was even increased by methylprednisolone in asthmatic airway mucosa, whereas eosinophil and CC chemokine expression was decreased [42]. These results reflect the current discussion about the dual nature of corticosteroids exerting differential effects on the regulation of chemokines. In addition, partial inhibition with prednisolone might also be explained by other chemokine-inducing factors such as lipoteichoic acids (LTA) which might also be present in staphylococcal supernatants.

In contrast to IL-8, GRO-α and ENA-78, no substantial quantities of GCP-2 protein were produced by A549 cells. To examine other proinflammatory stimuli that are capable of inducing GCP-2, we used IL-1β and LPS in a concentration-dependent manner [43] (results not shown). Even high concentrations of LPS did not lead to a significant release of GCP-2. IL-1β at a concentration of 10 ng/ml induced ENA-78, GRO-α, followed by IL-8 and GCP-2, which is in accordance with the results of other stimulation experiments in endothelial cells, human umbilical vein endothelial cells (HUVECs) and A549 cells [44]. Despite the fact that the production of GCP-2 by most cell types is weak in vitro[45], the detection of GCP-2 by immunohistochemistry is pronounced in the nasal epithelium, as it is in other forms of tissue [44]. By contrast, IL-8 is weakly detected by immunohistochemistry while it is produced at high levels in fibroblasts, endothelial cells and monocytes [45]. ENA-78 was rarely detectable in our immunohistochemistry, whereas abundant ENA-78 expression in A549 cells was expected, as it was originally cloned from this cell line [46]. The specific GCP-2 staining in the nasal epithelium suggests that the function of GCP-2 is different from that of IL-8 and ENA-78, despite their structural relationship. Our findings support the notion that the chemokine network shows complementarity rather than redundancy, as assumed by others [44].

To summarize, we demonstrated epithelial expression of CXC chemokines in chronic rhinosinusitis using immunohistochemistry. In a cell culture system of A549 cells, which are known to express PAR-1 to PAR-4, we showed PAR-2- and PAR-4-dependent stimulation of the neutrophil chemokines IL-8, GRO-α and ENA-78 by trypsin. Addition of the serine protease AEBSF to supernatants of different staphylococci revealed that soluble serine proteases are involved in chemokine regulation, and thus might activate PARs. In conclusion, bacterial serine proteases might be involved in neutrophilic airway inflammation in chronic rhinosinusitis.

References

1. Meltzer EO, Hamilos DL, Hadley JA, et al. Rhinosinusitis: establishing definitions for clinical research and patient care. J Allergy Clin Immunol. 2004;114:155–212. [PubMed]
2. Rudack C, Sachse F, Alberty J. Chronic rhinosinusitis − need for further classification? Inflamm Res. 2004;53:111–7. [PubMed]
3. Hsu J, Lanza DC, Kennedy DW. Antimicrobial resistance in bacterial chronic sinusitis. Am J Rhinol. 1998;12:243–8. [PubMed]
4. Biel MA, Brown CA, Levinson RM, et al. Evaluation of the microbiology of chronic maxillary sinusitis. Ann Otol Rhinol Laryngol. 1998;107:942–5. [PubMed]
5. Brook I. Sinusitis − overcoming bacterial resistance. Int J Pediatr Otorhinolaryngol. 2001;58:27–36. [PubMed]
6. Zhang N, Gevaert P, van Zele T, et al. An update on the impact of Staphylococcus aureus enterotoxins in chronic sinusitis with nasal polyposis. Rhinology. 2005;43:162–8. [PubMed]
7. Reed CE, Kita H.  The role of protease activation of inflammation in allergic respiratory diseases. J Allergy Clin Immunol. 2004;114:997–1008. [PubMed]
8. Ratner AJ, Lysenko ES, Paul MN, Weiser JN. Synergistic proinflammatory responses induced by polymicrobial colonization of epithelial surfaces. Proc Natl Acad Sci USA. 2005;102:3429–34. [PMC free article] [PubMed]
9. Asokananthan N, Graham PT, Fink J, et al. Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells. J Immunol. 2002;168:3577–85. [PubMed]
10. Becker K, Roth R, Peters G. Rapid and specific detection of toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene. J Clin Microbiol. 1998;36:2548–53. [PMC free article] [PubMed]
11. Jonsson IM, von Eiff C, Proctor RA, Peters G, Ryden C, Tarkowski A. Virulence of a hemB mutant displaying the phenotype of a Staphylococcus aureus small colony variant in a murine model of septic arthritis. Microb Pathog. 2003;34:73–9. [PubMed]
12. Becker K, Haverkamper G, von Eiff C, Roth R, Peters G. Survey of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene in non-Staphylococcus aureus species. Eur J Clin Microbiol Infect Dis. 2001;20:407–9. [PubMed]
13. Buddenkotte J, Stroh C, Engels IH, et al. Agonists of proteinase-activated receptor-2 stimulate upregulation of intercellular cell adhesion molecule-1 in primary human keratinocytes via activation of NF-kappa B. J Invest Dermatol. 2005;124:38–45. [PubMed]
14. Rudack C, Stoll W, Bachert C. Cytokines in nasal polyposis, acute and chronic sinusitis. Am J Rhinol. 1998;12:383–8. [PubMed]
15. Bachert C, Hormann K, Mosges R, et al. An update on the diagnosis and treatment of sinusitis and nasal polyposis. Allergy. 2003;58:176–91. [PubMed]
16. Watelet JB, Bachert C, Claeys C, van Cauwenberge P. Matrix metalloproteinases MMP-7, MMP-9 and their tissue inhibitor TIMP-1: expression in chronic sinusitis vs nasal polyposis. Allergy. 2004;59:54–60. [PubMed]
17. Gwaltney JM, Jr, Savolainen S, Rivas P, et al. Comparative effectiveness and safety of cefdinir and amoxicillin-clavulanate in treatment of acute community-acquired bacterial sinusitis. Antimicrob Agents Chemother. 1997;41:1517–20. Cefdinir Sinusitis Study Group. [PMC free article] [PubMed]
18. Doyle PW, Woodham JD. Microbiology and histopathology of chronic ethmoiditis. J Otolaryngol. 1991;20:445–7. [PubMed]
19. Hoyt WH., III Bacterial patterns found in surgery patients with chronic sinusitis. J Am Osteopath Assoc. 1992;92:1992. [PubMed]
20. Brook I, Frazier EH. Correlation between microbiology and previous sinus surgery in patients with chronic maxillary sinusitis. Ann Otol Rhinol Laryngol. 2001;110:148–51. [PubMed]
21. Finegold SM, Flynn MJ, Rose FV, et al. Bacteriologic findings associated with chronic bacterial maxillary sinusitis in adults. Clin Infect Dis. 2002;35:428–33. [PubMed]
22. Jiang RS, Lin PK, Lin JF. Correlation between bacteriology and computed tomography staging for chronic sinusitis. J Laryngol Otol. 2005;119:193–7. [PubMed]
23. von Eiff C, Peters G, Heilmann C. Pathogenesis of infections due to coagulase-negative staphylococci. Lancet Infect Dis. 2002;2:677–85. [PubMed]
24. Teufel P, Gotz F. Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis. J Bacteriol. 1993;175:4218–24. [PMC free article] [PubMed]
25. Lowy FD. Staphylococcus aureus infections. N Engl J Med. 1998;339:520–32. [PubMed]
26. Vaudaux P, Francois P, Bisognano C, et al. Increased expression of clumping factor and fibronectin-binding proteins by hemB mutants of Staphylococcus aureus expressing small colony variant phenotypes. Infect Immun. 2002;70:5428–37. [PMC free article] [PubMed]
27. Cheung AL, Bayer AS, Zhang G, Gresham H, Xiong YQ. Regulation of virulence determinants in vitro and in vivo in Staphylococcus aureus. FEMS Immunol Med. 2004;40:1–9. [PubMed]
28. Bronner S, Monteil H, Prevost G. Regulation of virulence determinants in Staphylococcus aureus: complexity and applications. FEMS Microbiol Rev. 2004;28:183–200. [PubMed]
29. Chan PF, Foster SJ. Role of SarA in virulence determinant production and environmental signal transduction in Staphylococcus aureus. J Bacteriol. 1998;180:6232–41. [PMC free article] [PubMed]
30. Reed SB, Wesson CA, Liou LE, et al. Molecular characterization of a novel Staphylococcus aureus serine protease operon. Infect Immun. 2001;69:1521–7. [PMC free article] [PubMed]
31. Dunman PM, Murphy E, Haney S, et al. Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. J Bacteriol. 2001;183:7341–53. [PMC free article] [PubMed]
32. Seiberling KA, Conley DB, Tripathi A, et al. Superantigens and chronic rhinosinusitis: detection of staphylococcal exotoxins in nasal polyps. Laryngoscope. 2005;115:1580–5. [PubMed]
33. Bernstein JM, Kansal R. Superantigen hypothesis for the early development of chronic hyperplastic sinusitis with massive nasal polyposis. Curr Opin Otolaryngol Head Neck Surg. 2005;13:39–44. [PubMed]
34. Kunkel SL, Standiford T, Kasahara K, Strieter RM. Interleukin-8 (IL-8): the major neutrophil chemotactic factor in the lung. Exp Lung Res. 1991;17:17–23. [PubMed]
35. DiMango E, Zar HJ, Bryan R, Prince A. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J Clin Invest. 1995;96:2204–10. [PMC free article] [PubMed]
36. Skerrett SJ, Liggitt HD, Hajjar AM, Ernst RK, Miller SI, Wilson CB. Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin. Am J Physiol Lung Cell Mol Physiol. 2004;287:L143–L152. [PubMed]
37. Dulon S, Cande C, Bunnett NW, Hollenberg MD, Chignard M, Pidard D. Proteinase-activated receptor-2 and human lung epithelial cells: disarming by neutrophil serine proteinases. Am J Respir Cell Mol Biol. 2003;28:339–46. [PubMed]
38. Page K, Hughes VS, Odoms KK, Dunsmore KE, Hershenson MB. German cockroach proteases regulate interleukin-8 expression via nuclear factor for interleukin-6 in human bronchial epithelial cells. Am J Respir Cell Mol Biol. 2005;32:225–31. [PubMed]
39. Knight DA, Lim S, Scaffidi AK, et al. Protease-activated receptors in human airways: upregulation of PAR-2 in respiratory epithelium from patients with asthma. J Allergy Clin Immunol. 2001;108:797–803. [PubMed]
40. Rudack C, Bachert C, Stoll W. Effect of prednisolone on cytokine synthesis in nasal polyps. J Interferon Cytokine Res. 1999;19:1031–5. [PubMed]
41. Schramm R, Thorlacius H. Staphylococcal enterotoxin B-induced acute inflammation is inhibited by dexamethasone: important role of CXC chemokines KC and macrophage inflammatory protein 2. Infect Immun. 2003;71:2542–7. [PMC free article] [PubMed]
42. Fukakusa M, Bergeron C, Tulic MK, et al. Oral corticosteroids decrease eosinophil and CC chemokine expression but increase neutrophil, IL-8, and IFN-gamma-inducible protein 10 expression in asthmatic airway mucosa. J Allergy Clin Immunol. 2005;115:280–6. [PubMed]
43. Froyen G, Proost P, Ronsse I, et al. Cloning, bacterial expression and biological characterization of recombinant human granulocyte chemotactic protein-2 and differential expression of granulocyte chemotactic protein-2 and epithelial cell-derived neutrophil activating peptide-78 mRNAs. Eur J Biochem. 1997;243:762–9. [PubMed]
44. Gijsbers K, Van Assche G, Joossens S, et al. CXCR1-binding chemokines in inflammatory bowel diseases: down-regulated IL-8/CXCL8 production by leukocytes in Crohn’s disease and selective GCP-2/CXCL6 expression in inflamed intestinal tissue. Eur J Immunol. 2004;34:1992–2000. [PubMed]
45. Wuyts A, Struyf S, Gijsbers K, et al. The CXC chemokine GCP-2/CXCL6 is predominantly induced in mesenchymal cells by interleukin-1beta and is down-regulated by interferon-gamma: comparison with interleukin-8/CXCL8. Lab Invest. 2003;83:23–34. [PubMed]
46. Walz A, Burgener R, Car B, Baggiolini M, Kunkel SL, Strieter RM. Structure and neutrophil-activating properties of a novel inflammatory peptide (ENA-78) with homology to interleukin 8. J Exp Med. 1991;174:1355–62. [PMC free article] [PubMed]

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