Induction of release and up-regulated gene expression of interleukin (IL)-8 in A549 cells by serine proteinases

Background Hypersecretion of cytokines and serine proteinases has been observed in asthma. Since protease-activated receptors (PARs) are receptors of several serine proteinases and airway epithelial cells are a major source of cytokines, the influence of serine proteinases and PARs on interleukin (IL)-8 secretion and gene expression in cultured A549 cells was examined. Results A549 cells express all four PARs at both protein and mRNA levels as assessed by flow cytometry, immunofluorescence microscopy and reverse transcription polymerase chain reaction (PCR). Thrombin, tryptase, elastase and trypsin induce a up to 8, 4.3, 4.4 and 5.1 fold increase in IL-8 release from A549 cells, respectively following 16 h incubation period. The thrombin, elastase and trypsin induced secretion of IL-8 can be abolished by their specific inhibitors. Agonist peptides of PAR-1, PAR-2 and PAR-4 stimulate up to 15.6, 6.6 and 3.5 fold increase in IL-8 secretion, respectively. Real time PCR shows that IL-8 mRNA is up-regulated by the serine proteinases tested and by agonist peptides of PAR-1 and PAR-2. Conclusion The proteinases, possibly through activation of PARs can stimulate IL-8 release from A549 cells, suggesting that they are likely to contribute to IL-8 related airway inflammatory disorders in man.


Background
Respiratory epithelium acts as the first tissue to meet inhaled pathogens and is capable of releasing inflammatory mediators and cytokines in response. Respiratory epithelial cells can synthesize and secrete a variety of proinflammatory cytokines such as IL-8, IL-1, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF) [1] and RANTES [2] which regulate cell behavior including growth, secretion, migration in physiological and pathological conditions. The importance of serine proteinases in the development of airway diseases has been emphasized in recent years. Of particular importance is that the potential role of tryptase [3] thrombin [4] and elastase [5] in the development of asthma, in which these serine proteinases were not only been over-secreted [4,6,7], but also found to play a role in induction of cytokine hypersecretion in airways [8,9]. However, the potential mechanism, through which these serine proteinases carry out their actions in respiratory tract, remains unclear. Since increased level of IL-8 in the airways reported to be closely correlated to asthma [10], we investigated the effect of tryptase, thrombin, trypsin, and elastase on IL-8 secretion and gene expression in A549 cells, a type II alveolar epithelial cell line from human adenocarcinoma, in the present study.
In recent years, PARs have been identified as receptors for serine proteinases. Among them, PAR-1 is a receptor of thrombin and trypsin [11]; PAR-2 is a receptor of trypsin, tryptase [12] and elastase [9]; PAR-3 [13] and PAR-4 [14] are receptors of thrombin. Activation of PARs could profoundly alter secretion ability of numerous cell types such as histamine release from human mast cells [15], IL-6 release from airway epithelial cells [8], IL-1 release from fibroblast [16], and IL-8 release from human oral epithelial cells [17]. We therefore investigated the effect of the agonists of all four types of PARs on IL-8 release from A549 cells in the current study. Since expression of PARs on A549 cells is crucial for the understanding of actions of the serine proteinases tested, we also investigated the expression PAR-1, PAR-2, PAR-3 and PAR-4 on A549 cells with immunocytochemical techniques and reverse transcription polymerase chain reaction (RT-PCR) in the present study.

Induction of IL-8 release by serine proteinases
Thrombin at concentrations of 1-10 U/ml provokes a concentration dependent release of IL-8 from A549 cells following 16 h incubation period. Approximately 8 fold increase in IL-8 release is observed at 16 h following incubation with 10 U/ml thrombin ( Figure 1A). The time course study shows that increased release of IL-8 induced by thrombin begins within 2 h, and lasts at least to 16 h ( Figure 1B).
At the concentrations from 1 to 300 ng/ml, trypsin is able to stimulate a 'bell shape' release of IL-8 from A549 cells following 16 h incubation period. The maximum release of 5.1 fold is observed when 3 ng/ml of trypsin was added to A549 cells. At the time of 8 h, however, a dose dependent release of IL-8 from A549 cells is achieved with 100 and 300 ng/ml trypsin. Small but nevertheless significant release of IL-8 is also observed with 300 ng/ml trypsin following 2 h incubation ( Figure 2). Also in Figure 2, it is clearly observed that the basal accumulated secretion of IL-8 from A549 cells is time dependent with 2.7 ± 0.7, 173 ± 54 and 329 ± 91 pg/ml being secreted following 2, 8 and 16 h incubation periods, respectively. Trypsin at concentration of 300 ng/ml fails to stimulate IL-10, IL-16, IL-17 and IL-18 secretion from A549 cells following 8 h incubation period (data not shown).
Tryptase at the concentrations from 0.125 to 2 µg/ml induces a concentration dependent IL-8 secretion from A549 cells. Approximately 4.3 fold increase in release of IL-8 is observed when 2 µg/ml of tryptase was incubated with cells for 16 h, and as little as 0.25 µg/ml tryptase is able to provoke a significant release of IL-8 from A549 cell at 16 h following incubation ( Figure 3A). Time course study reveals that increased release of IL-8 induced by tryptase begins within 2 h, and lasts at least to 16 h ( Figure  3B). Elastase, however, only at the concentrations of 0.1 and 0.3 µg/ml elicits significant release of IL-8 following 16 h incubation period, and the quantity of IL-8 released from A549 cells in response to 0.3 µg/ml elastase is similar to that induced by 2 µg/ml of tryptase ( Figure 3A). Time course study shows that elastase induced release of IL-8 occurs after 8 h incubation and maintains at least to 16 h ( Figure 3B).
Effect of thrombin on the release of IL-8 from A549 cells Figure 1 Effect of thrombin on the release of IL-8 from A549 cells. Cells were incubated (A) with various concentrations of thrombin at 37°C for 16 h, or (B) with 10 U/ml of thrombin for 2 h, 8 h and 16 h. Values shown are mean ± SE for 5 separate experiments. *P < 0.05 compared with the response to medium alone control.

Inhibition of IL-8 release induced by proteinases by their inhibitors
Hirudin, a specific thrombin inhibitor is able to inhibit thrombin-induced secretion of IL-8 at both 2 and 16 h following incubation. The maximum inhibition of approximately 89% is observed when 10 U/ml of hirudin was added to cells for 16 h (Table 2). Similarly, specific trypsin inhibitors SBTI and α 1 -antitrysin are able to completely abolish trypsin-induced secretion of IL-8 at both 8 and 16 h following incubation (Table 3). It is observed also that MSACK, an inhibitor of elastase completely abrogates elastase induced release of IL-8 (Table 4). In contrast, inhibitors of tryptase, benzamine and leupeptine are only able to inhibit tryptase induced IL-8 secretion by 47.5% and 6.5%, respectively following 16 h incubation (Table  4). Hirudin (Table 2), SBTI, and α 1 -antitrysin (Table 3), benzamidine, leupeptine and MSACK (Table 4) alone at the concentrations tested have little effect on IL-8 secretion from A549 cells.

Expression of PARs by A549 cells
FACS analysis shows that A549 cells express all four PARs regardless they are permeabilized or not ( Figure 4A). Immunofluorescent cell staining shows that PAR-2 seems mainly stained on the membrane surface of A549 cells, whereas PAR-1, PAR-3 and particularly PAR-4 predominately stained in cytoplasm ( Figure 4B). An agarose gel electrophoresis revealed that A549 cells express mRNAs for all four PARs ( Figure 4C). The amplified RT-PCR products of PAR-1, PAR-2, PAR-3 and PAR-4 mRNAs were sequenced and they all correspond to published sequences of PAR genes (data not shown).

Induction of IL-8 release by agonists of PARs
SFLLR-NH 2 , a specific PAR-1 agonist peptide stimulats a concentration dependent secretion of IL-8 from A549 cells following 16 h incubation ( Figure 5A), whereas its reverse peptide RLLFS-NH 2 has no effect on IL-8 release. The maximum release of IL-8 is 15.6 fold induced by 300 Effect of trypsin on the release of IL-8 from A549 cells µM of SFLLR-NH 2 following 16 h incubation period (Figure 5A). However, TFRGAP-NH 2 , an agonist peptide of PAR-3 and its reverse peptide PAGRFT-NH 2 at concentrations 0.1, 1, 10 and 100 µM do not show any influence on IL-8 release from A549 cells following 16 h incubation period (data not shown). The time course study shows that SFLLR-NH 2 induced release of IL-8 occurs after 8 h incubation and sustains at least until 16 h ( Figure 5B).
While SLIGKV-NH 2 and tc-LIGRLO-NH 2 , two specific agonists of PAR-2 induce concentration dependent secretion of IL-8 from A549 cells following 8 and 16 h incubation periods ( Figure 6B,6C) only tc-LIGRLO-NH 2 is able to stimulate IL-8 release at 2 h ( Figure 6A). The maximum release of IL-8 is approximately 79 and 6.6 fold over baseline induced by 100 µM of tc-LIGRLO-NH 2 at 2 h and 100 µM of SLIGKV-NH 2 at 8 h, respectively. VKGILS-NH 2 has little effect on IL-8 release, but tc-OLRGIL-NH 2 appears to induce a significant release IL-8 from A549 cells. However, the extent of release of IL-8 induced by tc-OLRGIL-NH 2 is much less than that induced by tc-LIGRLO-NH 2 ( Figure 6). SLIGKV-NH 2 and tc-LIGRLO-NH 2 at concentration of 10 µM fail to stimulate IL-10, IL-16, IL-17, and IL-18 secretion from A549 cells following 8 h incubation period (data not shown).
At a concentration of 10 µM, GYPGQV-NH 2 , an agonist peptide of PAR-4 induces a 3.5 fold increase in IL-8 release from A549 cells. However, at a higher concentration (100 µM), it stimulates less IL-8 secretion ( Figure 7A). VQG-PYG-NH 2 , a reverse peptide of GYPGQV-NH 2 , at the concentrations tested does not show any influence on IL-8 release ( Figure 7A). The time course study shows that GYPGQV-NH 2 induced release of IL-8 occurs after 8 h incubation and sustains at least until 16 h ( Figure 7B).

Effect of serine proteinases and agonists of PARs on expression of IL-8 mRNA in A549 cells
Thrombin, trypsin, tryptase and elastase stimulate an increase in expression of IL-8 mRNA in A549 cells when they were incubated with the cells. However, the tryptase induced up-regulation of expression of IL-8 mRNA only lasts for 2 h, whereas thrombin, trypsin (declined after 8 h) and elastase provoked expression of IL-8 mRNA continues until 16 h. Up to 6.8, 22.3, 9.9 and 7.8 fold increase in expression of IL-8 mRNA is observed with thrombin, trypsin, tryptase and elastase, respectively following incubation with A549 cells (Figure 8). Dramatically enhanced expression of IL-8 mRNA is found when SFLLR-NH 2 , SLIGKV-NH 2 or tc-LIGRLO-NH 2 was incubated with A549 cells for 2 h. At 8 and 16 h following incubation, however, IL-8 mRNA expression induced by SFLLR-NH 2 , SLIGKV-NH 2 or tc-LIGRLO-NH 2 is greatly decreased (Figure 8). GYPGQV-NH 2 and TFRGAP-NH 2 at 100 µM has little influence on IL-8 mRNA expression in A549 cells ( Figure   8). At the concentration of 100 µM, RLLFS-NH 2 , VKGILS-NH 2 , tc-OLRGIL-NH 2 , PAGRFT-NH 2 and VQGPYG-NH 2 , the reverse peptides of agonists of PARs have little effect on IL-8 mRNA expression in A549 cells (data not shown).

Discussion
It is demonstrated that human serine proteinases including thrombin, tryptase, elastase and trypsin are potent stimuli of IL-8 secretion from A549 cells, which suggests that they are likely to play a role in IL-8 related airway inflammatory disorders such as asthma, chronic obstructive pulmonary disease and cystic fibrosis.
As little as 5.6 nM of thrombin is able to stimulate approximately 2 fold increase in IL-8 secretion, and 56 nM of thrombin induces 8 fold increase in IL-8 release, indicating that this proteinase is a potent secretagogue of IL-8 release from A549 cells. Human mast cell tryptase, an established mediator of inflammation [18] at a concentration as low as 3.7 nM induces twice more IL-8 secretion over baseline release, and trypsin, a potential mediator of airway inflammation [19] at a concentration of 0.042 nM provokes approximately 3 fold increase in IL-8 secretion from A549 cells, suggesting that tryptic enzyme in airways may play a role in stimulation of IL-8 hypersecretion from airway epithelium. Similarly, elastase, a well-established mediator of airway inflammation at a concentration of 10.2 nM elicits 4.4-fold increase in IL-8 release, indicating that it is a potent secretagogue of IL-8 release from A549 cells as well. At a concentration of 345 nM, elastase was also found to be able to induce IL-8 and MCP-1 secretion from human gingival fibroblasts [9]. However, at the concentrations higher than 62.5 nM elastase could disarm PAR-2 within 10 min following incubation with human lung epithelial cells [20]. These findings suggest that elastase at lower concentrations induce cytokine release from A549 cells, but at higher concentrations may inactivate PAR-2 on human lung epithelial cells including A549 cells. It was impossible for us to examine the effect of elastase at the concentrations higher than 62.5 nM with our experimental system as at the concentration of 0.6 µg/ ml (20.4 nM), elastase was able to dissociate A549 cells from plate after 8 h incubation and the suspended cells died soon after (assessed by trypan blue staining). This phenomenon may explain the reason for which elastase at the concentration of 0.6 µg/ml fails to enhance IL-8 release. The similar phenomenon is also observed with trypsin at the concentrations higher than 1 µg/ml. These findings implicate that the detachment of bronchial epithelium observed in chronic airway inflammation may result from the hydrolytic activities of elastase and trypsin. Time course study shows that IL-8 release induced by thrombin, tryptase and trypsin initiates within 2 h following incubation, whereas the response to elastase occurs only after 8 h incubation period. This indicates that elastase and the other proteinases tested may adopt different mechanisms in induction of IL-8 release from A549 cells. The concentrations of tryptase and elastase used in the present study should be achievable under pathological conditions as the level of tryptase in asthmatic bronchial alveolar lavage fluid was 13.2 ng/ml [21] and the levels of elastase in asthmatic and cystic fibrosis sputum were 27 and 466 ng/ml, respectively [22]. While information on the levels of thrombin and trypsin in respiratory fluids are not available, a report described that trypsin-like activity was 46.9 mU/ml in mucoid sputum from patients with asthma [23] might implicate that the concentrations of thrombin and trypsin used in the current study ought to be achievable in the inflammatory airways.
A549 cells have been reported to secrete some 10 pg/ml of elafin and 3 ng/ml of secretory leukocyte protease inhibitor (SLPI) following 24 h incubation [24]. This concentration of elafin, an inhibitor of elastase should not affect the action of elastase on A549 cells, but the concentrations of SLPI, an inhibitor of trypsin and elastase may reduce the stimulatory action of the lower concentrations of trypsin or elastase on A549 cells.
Hirudin inhibites approximately 87% thrombin induced IL-8 secretion; SBTI and α 1 -antitrypsin completely abolish trypsin induced IL-8 secretion and MSACK eliminats 97% elastase induced IL-8 secretion, suggesting strongly that   the actions of these proteinases on A549 cells are dependent upon their intact catalytic sites. Since the known substrates of these proteinases on cells are PARs, the expression of PARs on A549 cells was investigated in the present study. To our surprise, benzamidine and leupeptine at a concentration of 30 µg/ml (a quite high concentration for the study on cells based on our previous work [15] are only able to inhibit tryptase induced IL-8 secretion by 47.5% and 6.5%, which suggests that IL-8 secretion induced by tryptase may not depend on its enzymatic activity, and there may be a receptor other than PARs being involved in the process. The similar findings on tryptase were observed previously in other studies [25]. However, to our knowledge, this is the first work examining the effects of thrombin, trypsin, tryptase and elastase on IL-8 release from airway epithelial cells under the same conditions.
It has been reported that a number of human cell types express more than one member of PAR family. Thus, platelets express PAR-1 and PAR-4 genes [14,26], endothelial cells express PAR-1, PAR-2, and possibly PAR-3 [13,27], fibroblast express PAR-1, PAR-2, PAR-3 and PAR-4 genes [28], smooth muscle cell express PAR-1, PAR-2, and PAR-3 genes [29] and respiratory epithelial cells express PAR-1, PAR-2, PAR-3 and PAR-4 genes and possibly proteins [8]. In the present study, we find that A549 cells express all four PARs at both protein and mRNA levels. Since expression of the PARs was observed under both permeabilized and non-permeabilized conditions, it is most likely that all these four PARs are located in both the cytoplasma and the plasma membrane surface of the cells.
SFLLR-NH 2 , tc-LIGRLO-NH 2 , SLIGKV-NH 2 and GYPGQV-NH 2 stimulates approximately 15.6, 79, 6.6, and 3.5 fold increase in release of IL-8, implicating that there are appropriate mechanisms to carry out IL-8 release process in response to PAR-1, PAR-2 and PAR-4 activation in A549 cells. However, A549 cells do not show any response (in terms of IL-8 release) to PAR-3 activation. Activation of A549 cells to release IL-8 by agonists of PARs indicates that the actions of thrombin, tryptase, elastase and trypsin on A549 cells are most likely carried out through hydrolytic cleavage of N-termini of PARs. The time course shows that the influence of agonists of PAR-1 and PAR-2 on A549 cells initiates within 2 h following incubation, but the action of agonist of PAR-4 on cells appears only after 8 h incubation. These observations suggest that the actions of thrombin on A549 cells are mainly (if not all) carried out through PAR-1, but not PAR-4, whereas the influence of trypsin on cells is most likely through both  PAR-1 and PAR-2. It is hard to understand the slower response of cells to elastase and at least partially enzymatic activity independent actions of tryptase on A549 cells. These obviously require further investigation. Using various concentrations of agonist peptides of PARs to stimulate A549 cells may better reflect the actions of these peptides on the cells, which reinforces the previous finding [8]. Up-regulation of expression of IL-8 gene in A549 cells by thrombin, trypsin, tryptase, elastase, PAR-1 and PAR-2 agonist peptides indicates that IL-8 released from A549 cells induced by these stimuli is most likely being newly generated, rather than pre-stored in the cells. The observation that relatively small quantity of IL-8 was released during the first 2 h of incubation in response to the above stimuli also supports our view. While the influence of tryptase and trypsin on IL-8 gene expression does not appear to have been studied previously, the report which found elastase [36,37] and thrombin [38] up-regulated IL-8 gene expression in human epithelial cells may support our current findings. To our knowledge, this is the first work examining IL-8 gene expression in response to sev-eral serine proteinases in epithelial cells under the same conditions. The parallel investigation of the actions of serine proteinases on A549 cells may contribute to easier understanding of the role of these proteinases in regulation of IL-8 gene expression. It is difficult to understand the reason why GYPGQV-NH 2 does not significantly upregulate IL-8 gene expression, but stimulates IL-8 release from A549 cells at 16 h following incubation. It could be that the significantly increased IL-8 gene expression occurs between 8 and 16 h incubation period, but we did not examine it.

Analysis of expression of PARs on A549 cells by flow cytometry (A), Immunofluorescent microscopy (B) and RT-PCR (C)
Effect of PAR-2 agonist peptides tc-LIGRLO-NH 2 and SLIGKV-NH 2 and their reverse peptides, tc-OLRGIL and VKGILS-NH 2 on IL-8 release from A549 cells Effect of SFLLR-NH 2 , an agonist peptide of PAR-1 and its reverse peptide RLLFS-NH 2 on IL-8 release from A549 cells Values shown are Mean ± SE for five separate experiments performed in duplicate. *P < 0.05 compared with the response to medium alone control; † P < 0.05 compared with the response to RLLFS-NH 2 at the same concentration.

SFLLR-NH 2
Induction of inflammatory mediator release from airway epithelial cells by agonists of PARs has been demonstrated previously. Thus, agonist of PAR-1 stimulated plateletderived growth factor secretion from lung epithelial cells [39]; agonists of PAR-2 stimulated IL-8 secretion from 16 HBE cells [40], GM-CSF and eotaxin release from human pulmonary epithelial cells [41] and matrix metalloproteinase-9 release from A549 and primary cultured small airway epithelial cells [42], and agonist of PAR-4 stimulated IL-8 secretion from human respiratory epithelial cells [8].
Our findings further strengthen the view that through activation of PARs, serine proteinases are actively involved in the pathogenesis of airway inflammation.
However, since A549 cells is not a normal airway epithelial cells, it may not fully represent the events happening in normal airway epithelial cells in response to the stimuli above in real life.

Conclusion
serine proteinases tested are potent stimuli of IL-8 secretion from A549 cells, and the influence of these proteinases on airway epithelial cells are most likely through activation of PARs. Induction of IL-8 secretion by proteinases indicates that they are likely to contribute to the pathogenesis of airway inflammatory disorders.
Development of proteinase inhibitor drugs may be valuable for treatment of these diseases.

Identification of expression of mRNA of PARs
The expression of mRNA of PARs by A549 cells was investigated with RT-PCR technique. Total RNA was isolated using TRIzol reagent according to the manufacturer's instructions. Briefly, cells were lysed directly by adding 1 ml of TRIzol Reagent to a 3.5 cm diameter dish (1 ml per 10 cm 2 ). A total of 200 µl of chloroform was added, and tubes were then centrifuged at 12,000 g for 15 min at 4°C, after which the aqueous phase was transferred to new tubes, RNA was precipitated by adding 0.5 ml of isopropyl alcohol, and then centrifuged at 12,000 g for 10 min at 4°C. Finally, 1 ml of 75% (v/v) ethanol was added to the pelleted RNA, and centrifuged at 7,500 g for 5 min at 4°C. Total RNA was quantified by measuring absorbance ratios at 260/280 nm. cDNA was prepared by reverse transcriptase using a commercial RNA-PCR kit, and reactions were performed according to the manufacturer's instructions. For each reaction, 1 µg of total RNA was reversely transcribed using oligo-d (T) and PAR-4 RT primers according to the protocol. The cDNA was amplified using forward and reverse specific primers for amplifying human PARs. β-actin was used as an internal control. Primers for PAR-1, PAR-2 and PAR-3 were designed based on PAR sequences in Genbank using Omiga software; Primer for PAR-4 was designed as described by Kahn et al [26]. Primers were prepared by Invitrogen Biotechnology Co., Ltd. The primer sequences were summarized in Table  1. The conditions for amplification were: for PAR-1, PAR-2, and PAR-3, the PCR mixture was heated at 94°C for 2 min followed by 35 cycles at 94°C for 30 sec, 67°C for 30 sec, 72°C for 1 min and 72°C for 10 min for 1 cycle; for PAR-4 and β-actin, the PCR mixture was heated at 94°C for 2 min followed by 35 cycles at 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec and 72°C for 10 min for 1 cycle. Electrophoresis was conducted in 1.5% agarose gels that were stained with SYBR Green I Nucleic Acid Gel Stain and photographed under UV light. PCR products were then sequenced.

Quantitative real-time PCR
IL-8 mRNA expression in A549 cells was determined by real-time PCR following the manufacture's protocol. Briefly, total RNA was isolated from the stimulated A549 cells using TRIzol Reagent. cDNA was synthesized from 5 µg of total RNA by using Superscript first strand synthesis system for RT-PCR and oligo-dT primers. A doublestranded DNA binding dye method was used for quantitative PCR/RT-PCR. Real-time PCR was performed in the ABI Prism 7700 Sequence Detection System (Perkin Elmer