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Am J Physiol Lung Cell Mol Physiol 293: L1208-L1218, 2007. First published August 31, 2007; doi:10.1152/ajplung.00137.2007
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PAR-2 activation and LPS synergistically enhance inflammatory signaling in airway epithelial cells by raising PAR expression level and interleukin-8 release

Ewa Ostrowska, Elena Sokolova, and Georg Reiser

Otto-von-Guericke-Universität Magdeburg, Medizinische Fakultät, Institut für Neurobiochemie, Magdeburg, Germany

Submitted 5 April 2007 ; accepted in final form 26 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Protease-activated receptors (PARs) are involved in the contribution of airway epithelial cells to the development of inflammation by release of pro- and anti-inflammatory mediators. Here, we evaluated in epithelial cells the influence of LPS and continuous PAR activation on PAR expression level and the release of the proinflammatory chemokine IL-8. We studied primary human small airway epithelial cells and two airway epithelial cell lines, A549 and HBE cells. LPS specifically upregulated expression of PAR-2 but not of PAR-1. Exposure of epithelial cells to PAR-1 or PAR-2 agonists increased the PAR-1 expression level. The PAR-2 agonist exhibited higher potency than PAR-1 activators. However, the combined exposure of epithelial cells to LPS and PAR agonists abrogated the PAR-1 upregulation. The PAR-2 expression level was also upregulated after exposure to PAR-1 or PAR-2 agonists. This elevation was higher than the effect of PAR agonists on the PAR-1 level. In contrast to the PAR-1 level, the PAR-2 level remained elevated under concomitant stimulation with LPS and PAR-2 agonist. Furthermore, activation of PAR-2, but not of PAR-1, caused production of IL-8 from the epithelial cells. Interestingly, both in the epithelial cell line and in primary epithelial cells, there was a potentiation of the stimulation of the IL-8 synthesis and release by PAR-2 agonist together with LPS. In summary, these results underline the important role of PAR-2 in human lung epithelial cells. Moreover, our study shows an intricate interplay between LPS and PAR agonists in affecting PAR regulation and IL-8 production.

protease-activated receptors; lipopolysaccharide; inflammatory mediators

IN ADDITION to cells of the immune system, the respiratory epithelium also actively participates in acute and chronic inflammatory reactions (6). Exposure of epithelial cells to deleterious agents triggers defense mechanisms and secretion of pro- and anti-inflammatory mediators. These can be allergens, bacteria, pollutants, and endogenous proinflammatory factors, such as early cytokines IL-1beta and TNF-{alpha}. Among the factors that are able to stimulate epithelial cells are also proteases of exogenous origin (from Aspergillus fumigatus, dust mites, bacteria Staphylococcus aureus, Pseudomonas aeruginosa) and endogenous origin (mast cell tryptase, neutrophil elastase, airway trypsin, thrombin). Those proteases react with the respiratory epithelium and cause immune responses by modulation of gene transcription and by synthesis and release of various mediators, such as cytokines, growth factors, prostanoids, leukotrienes, nitric oxide, and matrix metalloproteinases (MMPs) (3, 4, 10, 12, 32, 53).

Many of the cellular effects of proteases have been shown to be mediated via activation of protease-activated receptors (PARs) (52). PARs are widely distributed throughout the respiratory system. In the human airways, the presence of all four PARs (PAR-1 to -4) has been detected. PARs occur in the epithelium, smooth muscles, glands, fibroblasts, endothelium, and macrophages (40, 47, 58). Recent in vitro studies with cell lines clearly showed a strong impact of PAR activation in the lung epithelium on inflammatory responses and tissue remodeling. PARs on human primary epithelial cells or epithelial cell lines are responsible for the release of the cytoprotective and relaxant prostanoid prostaglandin (PG) E2, as well as release of proinflammatory cytokines IL-6 and IL-8 (2). PAR-2 activation induces production of eotaxin and granulocyte/macrophage colony-stimulating factor (59, 66). These factors promote eosinophil survival and recruitment. Epithelial PAR-2 has been shown to contribute to matrix remodeling by induction of release of MMP-9 (67).

Activation of PAR-2 has been documented to induce both pro- and anti-inflammatory in vivo effects in animal models of inflammation, including studies with PAR-2-deficient mice. PAR-2 activation inhibited the development of eosinophilia and induced bronchial relaxation (19, 48). PAR-2 was shown to be involved in airway hyperresponsiveness and eosinophil recruitment in allergen-challenged mice (21, 54, 61). However, PAR-2 itself does not initiate inflammatory cell influx, but enhances airway deleterious effects in challenged mice (21).

Some chronic lung diseases are frequently associated with increased PAR protein expression. For example, asthmatic bronchial epithelium showed significant increase in PAR-2 level compared with the nonasthmatic tissue (36). In preterm infants with prolonged chronic lung injury, PAR-2 immunoreactivity was significantly higher compared with newborn infants without pulmonary pathology (11). In animal models, LPS-induced inflammation and viral infection enhanced PAR levels and their responsiveness (31, 39, 48).

Studies on various cultured human airway cells showed different regulation of PAR expression by inflammatory mediators. In pulmonary artery endothelial cells, TNF-{alpha} induced the expression of PAR-2 but exerted no effect on PAR-1 level (24). IL-1beta upregulated PAR-2 in smooth muscle cells (23). In pulmonary fibroblasts, the profibrotic growth factors platelet-derived growth factor (PDGF) and transforming growth factor-beta1 stimulated PAR-2 expression (27). The antifibrotic and anti-inflammatory agent PGE2 was able to downregulate the expression of PAR-1, -2, and -3 (57).

However, there is still limited information concerning the question of whether PAR activation can modulate PAR expression on airway cells. This is an important issue for understanding lung pathology because elevated activity of potential PAR activators has been observed in airways with chronic inflammation. Among those proteases are thrombin (62), tryptase (7), and human airway trypsin-like protease (HAT) (45) as well as proteases from airborne allergens (32).

The investigation of the effect of persistent PAR activation by their agonists will provide important evidence for the involvement of PAR in many pathological conditions. Simultaneous occurrence of PAR activation and bacterial pathogen invasion represents an additional relevant issue for the better understanding of lung infectious and inflammatory diseases.

In the present study, we demonstrate that PAR-2 in lung epithelial cells is the major PAR type, which is upregulated upon PAR-1 and PAR-2 activation and LPS stimulation. We investigated the interplay between PAR agonists and the bacterial endotoxin in pulmonary epithelial cells. We demonstrate that simultaneous stimulation with LPS and PAR agonists abolished their individual stimulatory effects on PAR-1 expression, whereas PAR-2 expression remained elevated with simultaneous exposure to LPS and PAR agonists. Costimulation with PAR-2 agonist and LPS potentiated the production of the chemokine IL-8 compared with the stimulation by each agent alone. Our study reveals a new coordinated action of two known mediators of inflammation, PAR-2 and endotoxin, which contributes to the enhancement of inflammatory signaling in airways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Fura-2/AM was purchased from Molecular Probes (MoBiTec, Göttingen, Germany). The synthetic thrombin receptor agonist peptide (TRag; Ala-pFluoro-Phe-Arg-Cha-homoArg-Tyr-NH2) was from Neosystems Laboratoire (Strasbourg, France). Human PAR-2 activating peptide (SLIGKV-NH2) was from Bachem (Heidelberg), and PAR-3 activating peptide (TFRGAP-NH2) was synthesized by Biosynthan (Berlin, Germany). Thrombin and LPS from P. aeruginosa and Escherichia coli were from Sigma (Taufkirchen, Germany). Human IL-8 ELISA kits were from Amersham Biosciences (Freiburg, Germany). Antibodies against PAR-1 (WEDE15) were from Immunotech and against PAR-3 (H-103) were purchased from Santa Cruz (Heidelberg, Germany). Antibodies against PAR-2 (A5, polyclonal rabbit) were a generous gift from Dr. M. D. Hollenberg (Calgary, Canada). Alexa Fluor 488 goat anti-mouse Ig and goat anti-rabbit Ig antibodies were from Molecular Probes. The cell culture medium, DMEM, FCS, and antibiotics (penicillin and streptomycin) were from Biochrom (Berlin, Germany). Accutase was from PAA Laboratories (Coelbe, Germany).

Cell culture. A549 cells from American Type Culture Collection (Wesel, Germany) were cultured in DMEM supplemented with 10% FCS and 100 U/ml penicillin and 100 µg/ml streptomycin. The HBE cell line, kindly provided by Prof. Dr. L. Pott, Institut für Physiologie, Ruhr-Univesrsität Bochum, Germany, was cultured in DMEM Ham's F-12 (1:1) culture medium supplemented with gentamicin (50 µg/ml), kanamycin (50 µg/ml), insulin-transferrin-selenite (10 µg/ml), hydrocortisone (1 µM), pituitary extract (3.75 µg/ml), EGF (25 ng/ml), T3 (30 nM), and cholera toxin (10 ng/ml). Primary human small airway epithelial cells (HSAEC; Cambrex) (Verviers, Belgium) were grown in small airway cell basal medium supplemented with growth factors and antibiotics according to the manufacturer's instructions. Cells were kept at 37°C in a humidified atmosphere of 5% (vol/vol) CO2. Cells were passaged using Accutase to minimize the proteolytic activation of PARs.

Cytosolic Ca2+ measurement. The free intracellular Ca2+ concentration ([Ca2+]i) was measured using the Ca2+-sensitive fluorescent dye fura-2/AM. For dye loading, the cells grown on a coverslip were placed in 1 ml of HEPES-buffered saline (NaHBS, containing 20 mM HEPES, pH 7.4, 145 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 25 mM glucose) for 30 min at 37°C, supplemented with 2 µM fura-2/AM. Loaded cells were transferred into a perfusion chamber with a bath volume of ~0.2 ml and mounted on an inverted microscope (Axiovert 135; Zeiss, Jena, Germany). During the experiments, the cells were continuously superfused with NaHBS heated to 37°C.

Single cell fluorescence measurements of [Ca2+]i were performed using an imaging system from T.I.L.L. Photonics (Munich, Germany). Cells were excited alternately at 340 and 380 nm for 25–75 ms at each wavelength with a rate of 0.33 Hz, and the resultant emission was collected above 510 nm. Images were stored on a personal computer, and subsequently the changes in fluorescence ratio (F340 nm/F380 nm) were determined from selected regions of interest covering a single cell.

Real-time RT-PCR analysis. Total RNA was isolated from the cells with the RNeasy kit (Qiagen, Hilden, Germany). The isolation included DNase treatment. Reverse transcription was carried out with 1 µg of each RNA with iScript cDNA synthesis kit (Bio-Rad) in a final volume of 20 µl according to the manufacturer's protocol. Real-time PCR was performed on the iCycler (Bio-Rad) in 25 µl of reaction volume using SYBR green PCR Master Mix (Bio-Rad), as described by the manufacturer. Primer sequence pairs are listed in Table 1. The usage of intron-flanking primers, additionally with DNase treatment during RNA isolation, excludes the possibility of genomic DNA amplification. The thermal cycling conditions included denaturation step at 95°C for 3 min, followed by 40 cycles at 94°C for 30 s, 60°C (PAR-1-3, IL-8, and GAPDH) or 64°C (PAR-4) for 90 s, 72°C for 1 min, and the final melting curve program with ramping rate of 0.5°C/s from 60°C to 95°C. Amplification specificity of PCR products was confirmed by melting curve analysis and agarose gel electrophoresis. All mRNA measurements were normalized to the GAPDH mRNA level, which was unchanged in control and treated cells.


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  		Table 1. PCR primer pairs for human PAR-1, PAR-2, PAR-3, PAR-4, IL-8, and GAPDH

 
Immunocytochemistry. A549 cells grown on coverslips were washed three times in PBS and incubated with primary antibodies against PAR-1 and PAR-3 (20 µg/ml) and with rabbit antiserum against PAR-2 (dilution 1:50) at 4°C for 1 h. Cells were then washed three times in PBS and incubated with secondary antibodies conjugated to Alexa Fluor 488 (5–10 µg/ml) at 4°C for 1 h. Controls were made by omitting the primary antibodies. The negative controls in this study yielded no detectable labeling. The specificity of anti-PAR-2 antibodies was confirmed by the research group of Dr. M. D. Hollenberg (as documented in Ref. 9). The cells were examined with an LSM510 confocal laser scanning microscope (Carl Zeiss, Germany).

Analysis of fluorescence intensities. Fluorescence intensities of confocal images were analyzed using the Zeiss LSM 510meta software histomacro. Regions of interest (closed free-shape curves surrounding ca. 60 µm2) were set on the membrane of single cells, and the average fluorescence intensity in the region of interest (ROI) was determined. The intensity values for the treated cells were normalized to untreated cells, which were set as 100%.

IL-8 protein detection. According to the manufacturers' protocols, IL-8 protein levels were determined using human IL-8 ELISA kits. Briefly, serum-starved A549 cells were stimulated with TRag, thrombin, and PAR-2 AP (activating peptide) alone or together with LPS for 24 h, and then supernatants were collected for ELISA analysis. The levels of IL-8 were assayed at an optical density (OD) of 450 nm.

Statistical analysis. Statistical evaluation was carried out by t-test and multiple comparisons by one-way ANOVA with Dunnett's correction with P < 0.05 considered as significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression levels of PARs and functional activity of receptors in human respiratory epithelial cells. The presence of PAR subtypes on epithelial cell lines A549 and 16HBE as well as on primary cultures of HSAEC was determined by RT-PCR and immunocytochemistry. We confirmed our previous observations that A549 cells express three of the four PAR subtypes: PAR-1, PAR-2, and PAR-3, whereas HBE cells express PAR-1 and PAR-2 (26, 65). In HSAEC, we could determine the expression of PAR-1 and PAR-2. Quantification of the relative abundance of PARs showed the predominance of PAR-2 mRNA in all respiratory epithelial cells tested (Fig. 1A). PAR-1 expression levels were seven, twelve, and six times less than the PAR-2 transcript level in A549, HBE cells, and HSAEC, respectively. PAR-3 expression level in A549 cells was comparable to the PAR-1 transcript level. Immunostaining with antibodies against NH2-terminal parts of PAR-1, PAR-2, and PAR-3 confirmed the presence of these three PAR proteins on the plasma membrane of A549 cells (Fig. 1B, top), PAR-1 and PAR-2 in HSAEC (Fig. 1B, bottom), and in HBE cells (data not shown). The fluorescence staining for PAR-1 was always weaker than that for PAR-2 and was hardly detectable on some portions of the cells. This is consistent with the low transcript level of PAR-1.


Figure 1
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  		Fig. 1. Expression and immunofluorescence staining of proteinase-activated receptors (PARs) in human airway epithelial cells. A: PAR expression levels were determined in human A549, HBE, and primary human small airway epithelial cells (HSAEC) by real-time PCR. PAR mRNA expression was calculated using GAPDH as a reference gene and is expressed relative to the PAR-1 mRNA expression in A549 cells, which was chosen as a reference value of 1. Data are means ± SE of 3 independent experiments. B: A549 cells (top) and HSAEC (bottom) were incubated with antibodies directed against PAR-1, PAR-2, and PAR-3. PAR localization was visualized by staining with secondary antibodies conjugated with the fluorescent dye Alexa Fluor 488. Images are representative for 3 different experiments.

 
To confirm the functional expression of PARs, we examined the effect of PAR agonists on [Ca2+]i. Short-term application of PAR-1 agonists, the synthetic PAR-1-activating peptide TRag (5 µM), and thrombin (5 U/ml), induced a transient increase of [Ca2+]i in the A549 cell line (Fig. 2, A and C) and primary HSAEC (Fig. 2, B and D) and comparably in the HBE cell line (data not shown). All three cell lines demonstrated high heterogeneity in terms of Ca2+ responses to TRag, even within the same experiment. The PAR-1-activating peptide induced a [Ca2+]i elevation in a small number of the cells, whereas the majority of the cells remained unresponsive. Moreover, only at concentrations of 5 µM and higher, TRag produced a Ca2+ response in epithelial cells. For comparison, in human lung fibroblasts, the EC50 value for TRag was 1 µM (57). This can be due to the low density of PAR-1 on the cell surface that was shown immunocytochemically, which is not sufficient for stable Ca2+ signaling. Similar to TRag, thrombin induced a heterogeneous Ca2+ response in HSAEC, whereas in A549 cells, an elevation of [Ca2+]i was observed for all cells. This may be due, in A549 cells, to the expression of another thrombin receptor, PAR-3. A transient elevation of [Ca2+]i was achieved also after application of the synthetic PAR-2-activating peptide (PAR-2 AP) SLIGKV-NH2 (200 µM) (Fig. 2, E and F). In contrast to PAR-1 AP, PAR-2 AP always induced stable Ca2+ responses in all the epithelial cells tested. This reflects the high level of PAR-2 expression. Application of the synthetic PAR-3-activating peptide (TRFGAP-NH2) to A549 cells did not induce any effect. However, it should be noted that the effectiveness and specificity of this peptide is still controversial (8, 29, 30, 33, 57, 68). Therefore, we can conclude that PAR-2 is the main functionally active receptor in lung epithelial cell lines A549, HBE, and primary HSAEC in terms of Ca2+ signaling.


Figure 2
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  		Fig. 2. PAR agonist-induced intracellular calcium ([Ca2+]i) responses in human airway epithelial cells. Ca2+ responses elicited in A549 cells (A, C, E) and primary HSAEC (B, D, F) by the PAR agonists thrombin receptor agonist (TRag), thrombin, and PAR-2 activating peptide (AP). The fura-2 AM-loaded cells were exposed to 5 µM TRag, 5 U/ml thrombin, or 200 µM PAR-2 AP for 60 s, as shown by the horizontal bar. The increase in [Ca2+]i is indicated by the change in the measured fluorescence ratio (F340/F380 nm). The traces give responses of individual cells from 1 experiment. The images are representative for at least 3 different experiments.

 
Effect of continuous PAR activation on PAR-2 mRNA expression level. By real-time PCR analysis, we examined the effect of continuous PAR activation on the expression of the most abundant PAR in epithelial cells, PAR-2. Stimulation with the PAR-1 agonist TRag (5 µM) for 4 h slightly upregulated the PAR-2 level up to 1.5-fold in A549 and HBE cells (Fig. 3). Longer stimulation (24 h) resulted in further, statistically significant upregulation of PAR-2 mRNA in both cell lines (2.0- and 1.6-fold for A549 and HBE cells, respectively, P < 0.01). In HSAEC, TRag did not change the PAR-2 level after short and long periods of incubation (Fig. 3).


Figure 3
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  		Fig. 3. Modulation of PAR-2 expression level by PAR agonists in A549 cells, HSAEC, and HBE cells. Changes in PAR-2 mRNA level after treatment with PAR-1 agonist (5 µM TRag) or with PAR-2 agonist (200 µM PAR-2 AP) for the times indicated in A549 cells, HSAEC, and HBE cells. Measurements were done by real-time PCR. All cell lines were grown to 70–80% of confluency, growth medium was replaced by serum-free medium (for A549 cells) or fresh medium (for HBE cells and HSAEC), and cells were incubated with the given agonists. Total RNA was isolated and used for real-time PCR. Modulation of mRNA expression was calculated using GAPDH as a reference gene. Data are means ± SE of 3 independent experiments. Dotted line at 1.0 indicates control level. Gray bars below the graphs indicate the agonist used. *P < 0.05, **P < 0.01, significant difference compared with the untreated cells.

 
Activation of PAR-2 by PAR-2 AP for 4 h significantly upregulated PAR-2 mRNA levels in A549 and HBE cells (2.7- and 1.6-fold, respectively, P < 0.01) and slightly in HSAEC (Fig. 3). After 24 h of stimulation with PAR-2 AP, the transcript level for PAR-2 in A549 cells tended to decline but was still significantly higher than the control level (P < 0.05). In HBE cells and HSAEC, the 24-h incubation with PAR-2 AP increased the PAR-2 transcript level even more than after the stimulation for 4 h (2.3- and 1.7-fold, P < 0.01 and P < 0.05, respectively).

Together, PAR-2 activation induced higher upregulation of PAR-2 expression in epithelial cells than activation of PAR-1.

Effect of LPS and the combination of LPS with PAR agonists on PAR expression. We examined the influence of inflammatory mediators on PAR-2 expression in epithelial cells. Exposure of A549 cells to LPS from P. aeruginosa (10 µg/ml) for 4 h induced significant upregulation of PAR-2 expression, ~1.8-fold above control (P < 0.01). After 24 h of incubation, the value returned to basal level (Fig. 4A). For comparison, LPS from E. coli (0111:B4), which was shown to be a less potent activator in A549 cells than LPS from P. aeruginosa (37), did not change the PAR-2 expression level (data not shown). TNF-{alpha} (20 ng/ml) induced upregulation of the PAR-2 transcript level only after the long-term stimulation of 24 h (1.7 fold; data not shown).


Figure 4
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  		Fig. 4. The influence of PAR agonists and LPS, as well as simultaneous action of PAR agonists and LPS, on PAR expression levels in A549 cells. Changes in PAR-2 (A), PAR-1 (B), and PAR-3 (C) mRNA level after stimulation with TRag, PAR-2 AP, or LPS alone and after simultaneous incubation with PAR agonist together with LPS. A549 cells were grown to 70–80% of confluency, then growth medium was replaced by serum-free medium, and cells were incubated with LPS (10 µg/ml), TRag (5 µM), or PAR-2 AP (200 µM) for the different times indicated. Total RNA was isolated and used for real-time PCR. Modulation of mRNA expression was calculated using GAPDH as a reference gene. Data are means ± SE of 3 independent experiments. Dotted line at 1.0 indicates control level. Gray bars below the graphs indicate application of LPS. *P < 0.05, **P < 0.01, ***P < 0.001, significant difference compared with the untreated cells.

 
Next, we studied how PAR expression in A549 cells might be affected by simultaneous treatment with LPS and the PAR agonists. This combination of stimuli may reflect the pathological situation in vivo when both proinflammatory mediators, e.g., from pathogens and PAR-activating proteases, are present in the airways at elevated levels. Incubation of A549 cells in the presence of LPS together with the PAR-1 agonists TRag (Fig. 4A) and thrombin (data not shown) for 4 and 24 h completely abolished the upregulation of PAR-2 induced by TRag alone or by LPS alone. After the stimulation of the cells with LPS together with PAR-2 AP, the PAR-2 expression level after 4 h of concomitant incubation remained elevated and was comparable to the effect of LPS alone (Fig. 4A).

We attempted to measure changes of PAR-2 protein level on the cell surface using immunocytochemical analysis. We stimulated epithelial cells with either LPS or PAR-2 AP or with their combination for 4 h and waited for an additional 2-h incubation period after withdrawal of the stimuli. Then we observed an increase by 30% in the fluorescence signal intensity of the PAR-2 (for quantification, see MATERIALS AND METHODS). These results correlate with the data obtained by RT-PCR.

We next sought to determine whether other PARs expressed in airway epithelial cells could be influenced by LPS, PAR agonists, and their combination. Stimulation with the PAR-1 agonist TRag for 4 h did not affect PAR-1 expression, whereas longer stimulation (24 h) resulted in significant upregulation of PAR-1 mRNA by 1.5-fold (P < 0.01; Fig. 4B). Activation of PAR-2 by PAR-2 AP for 4 h upregulated PAR-1 by 1.6-fold (P < 0.01). After 24 h of stimulation with PAR-2 AP, the transcript level for PAR-1 remained elevated. LPS alone did not affect the PAR-1 expression. However, LPS, when applied together with TRag or PAR-2 AP, completely abolished the effects of the PAR agonists on PAR-1 expression level (Fig. 4B).

All PAR agonists tested, as well as LPS, when applied alone, did not affect the PAR-3 expression level. Interestingly, prolonged simultaneous treatment (24 h) with LPS and the various PAR agonists resulted in downregulation of the PAR-3 mRNA level. The decrease was up to two times (P < 0.001 for the combination of PAR-2 AP and LPS) (Fig. 4C).

LPS potentiates the effects of PAR-2 AP and thrombin on IL-8 production by epithelial cells. We further found that in A549 and HBE cell lines as well as in primary HSAEC, activation of PAR-2, but not PAR-1, resulted in the production of the proinflammatory chemokine IL-8. Stimulation of A549 cells and HSAEC with PAR-1-activating peptide TRag did not induce IL-8 mRNA synthesis (Fig. 5, A and B) and protein release from the cells (Fig. 5, C and D). Similar results were obtained with HBE cells (data not shown). Stimulation of A549 cells and HSAEC with thrombin resulted in an increase of the IL-8 transcript level and in protein release (Fig. 5), whereas HBE cells were unresponsive to thrombin (data not shown). This discrepancy in response of different epithelial cell lines to thrombin can be explained by the ability of thrombin to possibly activate a receptor other than the PARs, which is expressed in the A549 cell line and HSAEC. We could consistently find the inability of PAR-1 to induce IL-8 production in lung epithelial cell lines.


Figure 5
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  		Fig. 5. Induction of IL-8 mRNA synthesis and protein release by PAR agonists, alone or in combination with LPS, from A549 cells and HSAEC. Changes in IL-8 mRNA level in A549 cells (A) and in HSAEC (B) after 4 and 24 h of incubation were detected by real-time PCR. Secreted IL-8 in the supernatant after 24 h of stimulation from A549 cells (C) and HSAEC (D) was quantified by ELISA. The cells were grown to 70–80% of confluency, growth medium was replaced by fresh medium (for HSAEC) or serum-free medium (for A549 cells), and cells were incubated with 5 µM TRag, 5 U/ml thrombin, or 200 µM PAR-2 AP alone or together with LPS (10 µg/ml). The results are means ± SE of 3 independent experiments. Dotted line at 1.0 indicates control level. Gray bars below the graphs indicate application of LPS. ***P < 0.001, **P < 0.01, *P < 0.05, compared with the untreated cells; +P < 0.05, +++P < 0.001 compared with the cells exposed to LPS alone; ###P < 0.001, #P < 0.05 compared the cells treated with TRag, thrombin, or PAR-2 AP alone.

 
Stimulation of A549 cells with the specific PAR-2 AP peptide increased the IL-8 mRNA level after 4 h by 14-fold (P < 0.01). After 24 h of stimulation, the IL-8 mRNA level declined but was still 2.4-fold above the control level (P < 0.01; Fig. 5A). In primary HSAEC and HBE cells, PAR-2 AP induced a smaller increase than in A549 cells. In HBE cells, the IL-8 mRNA level was significantly increased with a 2.7-fold rise (P < 0.01) after a 4-h incubation. The effect persisted for up to 24 h (2.9-fold, P < 0.05) (data not shown). In HSAEC, the IL-8 expression was increased by 1.6-fold after a 4-h stimulation and was further increased after a 24-h stimulation (2.2-fold, P < 0.01; Fig. 5B). The delayed response of HSAEC to PAR-2 agonist might be a characteristic feature of primary cells, which was also documented in another work (67).

The effect of the PAR-2 agonist on the IL-8 mRNA level was seen similarly on protein level. PAR-2 AP stimulation resulted in significant increase of IL-8 concentration in the supernatant culture medium of A549 cells (Fig. 5C), HSAEC (Fig. 5D), and HBE cells (data not shown) (~17-, 2.0-, and 2.5-fold, respectively). A549 cells exhibited a higher potency than HBE and HSAEC with regard to the amount of the cytokine released that was also documented before (14, 41).

We next investigated the effect of simultaneous treatment with LPS and PAR agonists on the expression and synthesis of IL-8. LPS alone induced significant upregulation of the chemokine expression in A549 cells and HSAEC (7.6- and 2.3-fold, respectively). Challenging the A549 cells and HSAEC for 4 h with PAR-2 AP or thrombin in the presence of LPS amplified the IL-8 expression (Fig. 5, A and B). The IL-8 expression reached the highest level after a combined stimulation with PAR-2 AP and LPS compared with the costimulation with LPS and thrombin or with each stimulus alone. The potentiation of the IL-8 expression by simultaneous stimulation with LPS and PAR agonists was more prominent in A549 cells than in HSAEC. Costimulation with LPS and TRag did not significantly alter the IL-8 expression level that was achieved by LPS alone in these cells. After 24 h of incubation, the IL-8 mRNA levels were still elevated, but the potentiating effects were not seen any more.

After 24 h of incubation of A549 cells and HSAEC with the PAR agonists PAR-2 AP and thrombin, in the presence of LPS, the release of IL-8 into the culture medium was also greatly potentiated (Fig. 5, C and D). The effect of simultaneous treatment of A549 cells with LPS and thrombin was 3.2 times higher than that achieved by LPS alone and 4.9 times higher than that induced by thrombin alone. In HSAEC, the IL-8 concentration in the cell culture medium was increased by 1.7 times after stimulation with thrombin together with LPS compared with the effects of each stimulus alone. TRag did not influence the effect of LPS on IL-8 release in both cell lines. Similar to the effects observed on transcript levels, the highest protein release was achieved when the cells were stimulated with PAR-2 AP together with LPS. After the combined stimulation of A549 cells with PAR-2 AP and LPS, the IL-8 protein level was 5.5 times higher than after stimulation by LPS alone and 1.6 times higher than after stimulation by PAR-2 AP alone. For HSAEC, the respective increase of the IL-8 concentration was 1.9- and 2.3-fold, respectively. The higher effect achieved by PAR-2 activation than by PAR-1 activation again emphasizes the importance of the epithelial PAR-2 in airway pathophysiology.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There is growing evidence that airway epithelial cells play a key role in host responses to infectious and allergic stimuli. Diseased conditions including acute lung injury and chronic inflammation cause activation of epithelial cells and coordinated expression of a variety of proinflammatory factors during the inflammatory response. The cells persistently express abnormally high levels of bioactive molecules and surface receptors and are thus able to maintain the ongoing inflammation. Among the factors involved in the regulation of epithelial responses to injury are PARs. In the present study, we investigated the modulation of PAR expression after exposure of cells to PAR activators, LPS, and the combination of PAR agonists and LPS. Additionally, we evaluated the IL-8 production from airway epithelial cells. The control of PAR-1 or PAR-2 expression or IL-8 synthesis exerted by PAR activation and/or LPS exposure, which we observed in epithelial cells, is schematically summarized in Fig. 6.


Figure 6
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  		Fig. 6. Scheme summarizing the effects of the exposure of lung epithelial cells to PAR agonists and LPS. Application of PAR agonists and LPS as a single stimulus as well as the combined application are marked on top of the scheme in boxes within dotted frames. The effects of the stimulation of the cells to these agonists, given as response 1 (PAR-1 expression), response 2 (PAR-2 expression), and response 3 (IL-8 expression/release) are presented below. The thickness of the arrows illustrates the strength of the response. The dashed arrows with an X portray the lack of influence of the respective agonist.

 
First, we characterized functional responses caused by PAR-1 and PAR-2 subtypes, which are expressed in human lung epithelial cells. From our in vitro studies using human primary airway epithelial cells and cell lines, we conclude that PAR-2 is the main functional receptor on these cells. Moreover, we showed that continuous activation of both PAR-1 and PAR-2 caused upregulation of expression mostly of PAR-2. Activation of PAR-2 resulted in higher upregulation of the receptor than activation of PAR-1.

It is important to evaluate the feedback influence of persisting PAR activation on PAR expression because numerous observations have demonstrated that the activity of proteases, potential activators of PARs, is increased in damaged or inflamed lung. Lung epithelium is a source of potent PAR-2 activators, i.e., trypsin and human airway trypsin-like protease (HAT) (16, 60). Active HAT is the predominant trypsin-like protease in the sputum of patients with chronic bronchitis and bronchial asthma (45), whereas trypsin is present in bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease (COPD) (51). Another well-recognized PAR-2 activator, mast cell tryptase, is present at elevated levels in asthmatic airways (6). Asthma is characterized by increased vascular leakage and accumulation of plasma proteins in airways. The presence of thrombin activity in asthmatic airways has been documented (62). The observations showing that many actions of PAR-2 on epithelial cells are mediated by the basolaterally expressed receptor (38, 69) are in agreement with the notion that endogenous PAR activators indeed affect the receptors located on this side. This is due to the fact that proteases, which originate from the circulation (thrombin and other coagulation proteases) or are released from cells residing in both epithelial and mesenchymal space (e.g., tryptase), would encounter at first the basolateral PAR-2 when they migrate towards the epithelium.

We also investigated the influence of LPS on PAR levels in epithelial cells. LPS or endotoxin, a cell wall component of gram-negative bacteria, is a common occupational air contaminant that causes both acute and chronic airflow obstruction, airway inflammation, and airway remodeling. In our study, we used LPS from P. aeruginosa, an important respiratory opportunistic pathogen. LPS is a marker of a number of respiratory chronic diseases associated with inflammation, such as asthma and cystic fibrosis (22, 46). Respiratory epithelial cells along with alveolar macrophages are a target for endotoxin. Airway epithelial cells are known to respond to P. aeruginosa LPS by enhanced expression of defensins and adhesion molecules and release of different chemokines and cytokines (25, 37, 44). Besides the release of the inflammatory agents from the cells, LPS can modulate cellular responsiveness to subsequent stimuli via alteration of the expression of cell-surface receptors (24, 48). In our study, we showed that LPS affected solely PAR-2 but not PAR-1 expression on airway epithelial cells. A similar selectivity of LPS towards upregulation of PAR-2 was seen for endothelial cells (49).

We further analyzed the combined effect of LPS together with PAR agonists on PAR levels on epithelial cells. The combination of PAR-1 or PAR-2 agonists with LPS abrogated the effects on PAR-1 expression during all periods of incubation. At the same time, the PAR-2 expression level remained upregulated after short stimulation with the combination of LPS and PAR-2 AP. Thus, the elevation of PAR-2 expression persists even under those conditions where the expression of PAR-1 decreases. This is in line with the observations made in animal models of lung injury that showed that rat bronchi after LPS treatment exhibited increased levels of PAR-2 on the epithelium, and, as a consequence, increased responsiveness to PAR-2 activation (48). Our data are in good agreement with the report by Knight and coworkers (36), where asthmatic respiratory epithelium showed increased expression of PAR-2, but not of other PARs. These data underline the importance of epithelial PAR-2 and PAR-2 activators during acute lung injury and chronic inflammation.

Lung exposure to LPS induces mainly neutrophil-dominated inflammatory responses (64). The rapid invasion of neutrophils is a result of production of chemotactic factors from activated macrophages and epithelial cells. The main chemoattractant for neutrophils is IL-8. The chemokine release from epithelial cells is stimulated by activation of Toll-like receptor 4 not only by its specific activator LPS (15) but also by other factors, e.g., neutrophil elastase (20). Activation of other receptors on epithelial cells, such as PARs (2), can also stimulate IL-8 release. The elevated level of IL-8 is a characteristic feature of chronic inflammatory diseases, such as asthma of neutrophilic phenotype (63) and cystic fibrosis (5). The IL-8 level is also raised in airways of patients with COPD (50).

In our work, using primary airway epithelial cells and epithelial cell lines, we found that PAR-2 agonists and LPS substantially potentiated each other in the mRNA synthesis and protein release of IL-8 from the cells. Therefore, we conclude that epithelial PAR-2 synergistically with LPS can intensify neutrophil recruitment during bacterial infection. In agreement with this, PAR-2-deficient mice, compared with wild-type animals, showed lowered leukocyte rolling and reduced content of myeloperoxidase, a neutrophil marker. This was seen after surgical trauma and LPS-induced damage, respectively (35, 43).

Similarly to our findings, enhancement of PAR-induced cellular response by proinflammatory mediators was documented in endothelial cells (13). Simultaneous treatment of HUVEC with LPS or TNF-{alpha} and PAR agonists amplified the IL-6 production possibly via enhanced activation of NF-{kappa}B. We speculate that the potentiation of IL-8 production, observed in our work, induced by the combination of PAR-2 AP and LPS, is also due to increased transcriptional activity of factors responsible for IL-8 transcription. IL-8 transcription can be regulated by several transcription factors, including AP-1, nuclear factor (NF)-{kappa}B, AP-2, and NF-IL-6 (56, 71). PAR-2 activation in airway epithelial cells can induce AP-1 and NF-{kappa}B transcriptional activity (1, 55). The same transcription factors can be activated upon LPS exposure (18, 28, 42). We propose that simultaneous application of PAR-2 agonist and LPS to epithelial cells synergistically increases transcriptional activity of both AP-1 and NF-{kappa}B and their binding to the IL-8 promoter.

Under normal conditions, the epithelial cells produce proinflammatory chemokines and anti-inflammatory molecules, e.g., surfactant proteins, in a highly balanced manner, and the cellular response to the exposure of inhaled LPS is dampened. On the contrary, asthmatic conditions or viral infection sensitize airways to noxious agents. This is characterized by airway hyperresponsiveness. Disbalanced overproduction of proinflammatory mediators exacerbates the inflammatory airway status. The potentiation of IL-8 release by simultaneous activation of PAR-2 and exposure to LPS, as observed in our work, with intensification of neutrophil recruitment, is an example of the cooperative action of inflammatory mediators acting in chronic inflammatory airway diseases. Thus, the results of our study further support the proinflammatory role of PAR-2 in airways documented previously (21, 54, 61). The participation of PAR-2 in the amplification of inflammatory signaling underlines the enormous importance of this receptor in lung pathophysiology.

In our work, we found that only PAR-2 but not PAR-1 activation by the specific activating peptides leads to release of IL-8 and potentiates LPS-induced chemokine release from both primary epithelial cells and cell lines. The lack of influence of PAR-1 activation on IL-8 release in A549 cells appears to differ from the data of Asokananthan and coworkers (2). However, it should be noted that in their work, the effect of PAR-1 activation caused by the synthetic agonist peptide was very small compared with the effects caused by activation of the other PARs. The effect of thrombin on IL-8 release, which we observed in our work, can be due to activation of receptors for thrombin other than PAR-1.

In support of our observation of distinct roles of PARs in lung epithelium, several recent reports have shown that the different PAR subtypes elicit cellular responses, which are unique for each PAR. For example, in airway epithelial cells, PAR-2 but not PAR-1 activation resulted in PGE2 release, increased neutrophil adhesion to the cells, and changes in ion transport (17, 34, 70).

In summary, we demonstrate that among the PAR subtypes on airway epithelial cells, PAR-2 plays an exclusive role in terms of regulation of receptor expression and inflammatory responses. We demonstrate a cooperative action of PAR-2 and LPS in the production of the chemoattractant IL-8. These findings provide new insights into the role of epithelium in regulation of neutrophil recruitment.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from Sachsen-Anhalt (Program of Excellence 3521C/0703M and 3521A/0703M) and Bundesministerium für Bildung und Forschung (BMBF Grant 01ZZ0407).


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Z. Grishina for valuable suggestions and discussions and to Dr. F. Sedehizade for significant contributions at different stages of the project. We also thank E. Busse and A. Schneider for technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Reiser, Otto-von-Guericke-Universität Magdeburg, Medizinische Fakultät, Institut für Neurobiochemie, Leipziger Strasse 44, D-39120, Magdeburg, Germany (e-mail: georg.reiser{at}med.ovgu.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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