25 July 2021
Bradykinin- and lipopolysaccharide-induced bradykinin B(2) receptor expression, interleukin 8 release and "nitrosative stress" in bronchial epithelial cells BEAS-2B: Role for neutrophils. / Ricciardolo FLM; Sorbello V; Benedetto S; Defilippi I; Sabatini F; Robotti A; van Renswouw DC; Bucca C; Folkerts G; De Rose V.. - In: EUROPEAN JOURNAL OF
PHARMACOLOGY. - ISSN 0014-2999. - 694:1-3(2012), pp. 30-38. Original Citation:
Bradykinin- and lipopolysaccharide-induced bradykinin B(2) receptor expression, interleukin 8 release and "nitrosative stress" in bronchial epithelial cells BEAS-2B: Role for neutrophils.
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DOI:10.1016/j.ejphar.2012.07.051 Terms of use:
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J Pharmacol. 2012 Nov 5;694(1-3):30-8. doi: 10.1016/j.ejphar.2012.07.051.
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Bradykinin- and Lipopolysaccharide-induced bradykinin B
2receptor
expression, interleukin 8 release and “nitrosative stress” in bronchial epithelial
cells BEAS-2B: role for neutrophils
Fabio L.M. Ricciardoloa,, Valentina Sorbelloa, Sabrina Benedettoa, Ilaria Defilippia, Federica Sabatinib, Andrea Robottia, Dein C. van Renswouwc, Caterina Buccad, Gert Folkertsc, Virginia De Rosea.
a
Department of Clinical and Biological Sciences, Division of Respiratory Disease, University of Torino, Torino, Italy
b
Clinical Pathology Laboratories, Department of Experimental and Laboratory Medicine, G. Gaslini Institute, Genova, Italy
c
Department of Pharmacology and Pathophysiology, Faculty of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
d
Department of Clinical Pathophysiology, Respiratory Pathophysiology Unit, University of Torino, Torino, Italy
Corresponding author:
Dr. Fabio L.M. Ricciardolo, University of Torino
Dept. of Clinical and Biological Sciences, Division of Respiratory Disease San Luigi Gonzaga Hospital, Regione Gonzole 10
10043 Orbassano (Torino), Italy Phone: +39 011 9026777
Fax: +39 011 9026028
ABSTRACT
Bradykinin-induced interleukin (IL)-8 release should potentially activate neutrophils releasing myeloperoxidase (MPO) and subsequently generating “nitrosative stress”.
We studied bradykinin-induced expression of bradykinin B2 receptor and bradykinin- and
lipopolysaccharide (LPS)-induced IL-8 release, MPO (marker of neutrophil activation) and 3-nitrotyrosine (3-NT; marker of “nitrosative stress”) production in human bronchial epithelial cells BEAS-2B alone or in co-culture with human neutrophils.
We evaluated B2 receptor protein expression in BEAS-2B cells by immunostainings and Western
blot analysis, and measured respectively bradykinin- or LPS-induced IL-8 release in BEAS-2B cells and bradykinin- and/or LPS-induced MPO and 3-NT production in BEAS-2B cells co-cultured with human neutrophils by ELISA. In addition, we evaluated bradykinin- and/or LPS-induced 3-NT formation in BEAS-2B cells co-cultured with human neutrophils by immunocytochemistry.
Bradykinin up-regulates B2 receptor expression (P<0.05) and stimulate IL-8 release (P<0.001) in
BEAS-2B cells. Either the selective bradykinin B2 receptor antagonist HOE 140 or the selective
bradykinin B1 receptor antagonist Lys-(des-Arg9, Leu8)-bradykinin alone halved IL-8 release and
the combination of both drugs suppressed this effect. In BEAS-2B cells co-cultured with human neutrophils bradykinin increased MPO release and 3-NT production compared to BEAS-2B cells with human neutrophils (P<0.001), and the addition of LPS in BEAS-2B cells with human neutrophils and bradykinin induced a further dramatically increase of MPO release and 3-NT formation (P<0.001).
Bradykinin and LPS provoked “nitrosative stress”, potentially mediated by IL-8, in bronchial epithelium co-cultured with neutrophils suggesting a role for bradykinin in the amplification of chronic airway inflammation via production of “nitrosative stress”.
Keywords: bradykinin; bradykinin B2 receptor; nitrosative stress; interleukin-8; lipopolysaccharide; bronchial epithelium
1.
Introduction
The C-X-C chemokine interleukin (IL)-8 released by different structural cells, including airway epithelial cells, is a potent chemoattractant for neutrophils involved in airway inflammatory diseases (Rodgers et al., 2002). IL-8 promotes neutrophil chemotaxis contributing to host defence against pathogens as part of innate immunity; however, an amplification of this inflammatory response may contribute to the pathogenesis of chronic inflammatory disease (Rodgers et al., 2002; Malerba et al., 2006). Several agents potentially increase IL-8 release by airway epithelial cells, particularly cytokines [tumor necrosis factor (TNF)-α, interferon (IFN)-γ], respiratory viruses and bacterial products (Chmura et al., 2008; Newcomb et al., 2007; Massion et al., 1994).
Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria (Raetz and Whitfield, 2002), is released from bacteria during cell division or cell death. Upon its release, LPS binds to CD14, its primary receptor, mainly expressed on macrophages (Tobias and Ulevitch, 1993) and neutrophils (Wagner et al., 2003) increasing phagocytic activity and enhancing the secretion of pro-inflammatory cytokines such as TNF-α, IL-6, and others (Rosenfeld et al., 2006). The LPS-CD14 complex initiates intracellular signalling, by interacting with the transmembrane protein Toll-like receptor-4 (TLR-4), promoting the activation of nuclear factor–kappaB (NF-kB) transcription factor and extracellular signal-regulated kinase 1/2 (ERK1/2) (Jiang et al., 2000). Bradykinin, a nine-amino acid peptide, is formed in body fluids and tissues from the plasma precursor kininogen in response to infection and inflammatory stimuli (Abraham et al., 2006). Bradykinin induces its biological effects via two distinct mammalian bradykinin receptor subtypes: the constitutive B2 receptor and the inducible B1 receptor (Abraham et al., 2006). Bradykinin is
highly active on the B2 receptor and, to a lesser extent, also active on the B1 receptor with a lower
affinity than [des-Arg9]-bradykinin. Activation of B2 receptor, a G-protein-coupled receptor
constitutively expressed on most cell types, leads to a number of intracellular events (Greco et al., 2005). In contrast, B1 receptors are not present in tissues under normal conditions but their
expression can be induced during inflammation or tissue injury (Marceau, 1995). It has been reported that bradykinin concentration in bronchoalveolar lavage fluid of patients with chronic pulmonary diseases and pneumonia increases by about 5- to 10-fold (Baumgarten et al., 1992). Kinins exert a wide range of biological activities in the airways, including cytokines (IL-8), neuropeptides, prostaglandin PGE2 and NO release, vasodilatation, smooth muscle
contraction/relaxation, plasma exudation and leukocyte migration, most of which are mediated by bradykinin B2-receptors located on smooth muscle cells, epithelium, endothelium and also on
sensory nerves (Abraham et al., 2006, Bandeira-Melo et al., 1999).
A recent study showed that severe COPD patients have an exaggerated inflammatory response in the airways, characterised by neutrophil inflammation with polymorphonuclear neutrophils myeloperoxidase (MPO) over-expression and “nitrosative stress”: interaction between oxidative stress and NO metabolism with the production of reactive nitrogen species such as peroxynitrite which nitrates the tyrosine residues of proteins to yield the stable product 3-nitrotyrosine (Ricciardolo et al., 2005, 2006).
This study was designed to evaluate 1) the constitutive protein expression of B2 receptor in normal
human bronchial epithelial cells (BEAS-2B cells); 2) the effect of bradykinin on B2 receptor protein
expression in BEAS-2B cells; 3) the effect of bradykinin and LPS on IL-8 and MPO release and on 3-nitrotyrosine formation in BEAS-2B cells alone and in co-culture with human neutrophils.
2.
Materials and Methods
2.1. Cell culture
Immortalized, non-tumorigenic human bronchial epithelial cells BEAS 2B (CRL-9609; American Type Culture Collection, Manassan, VA) were cultured in a serum-free medium composed by 50% LHC9 medium (Gibco, Grand Island, NY, USA) and 50% RPMI-1640 medium (Euroclone Ltd, Paignton, UK) supplemented with 1X Non-Essential Amino Acids (Gibco), L-glutamine (2mM), penicillin (100 U/ml)/streptomycin (100 U/ml) (Euroclone Ltd) and incubated at 37°C in 100% humidity and 5% CO2.
Additionally, in some experiments polymorphonuclear neutrophils (PMNs) were co-cultured with BEAS-2B cells in 6-well plates (Falcon, BD Biosciences) equipped with porous cell culture membrane inserts with pore diameters of 1 μm (Falcon, BD Biosciences) (Blaheta et al., 2009).
2.2. Experimental study design
When 80% confluent, BEAS-2B cells were incubated in absence or presence of bradykinin acetate (Sigma Aldrich, St Louis, MO, USA) in order to perform immunostaining experiments and either a time-course (4, 8, 12 and 24 h; at 10-6M) or a dose-response curve (from 10-9M to 10-5M; at 12 h) for B2 receptor expression and IL-8 release. In some experiments, cells were incubated for 12 h with
Lipopolysaccharide (LPS) from Pseudomonas aeruginosa serotype 10 (Sigma Aldrich) (1 μg/μl) alone or in combination with bradykinin (10-6 M) and with bradykinin (10-6 M) alone.
In other experiments, concerning IL-8 release, BEAS-2B cells were also pre-treated for 1 h with the selective B2 receptor antagonist (D-Arg0, Hyp3, Thi5, D-Tic7, Oic8)-bradykinin (HOE 140) (Sigma
Aldrich) (1 M) or the selective B1 receptor antagonist Lys-(des-Arg9, Leu8)-bradykinin
trifluoroacetate salt (Sigma-Aldrich) (1 M) and incubated for 12 h in presence of bradykinin (10
-6
Furthermore, experiments to evaluate MPO and 3-nitrotyrosine (3-NT) production at different time points (baseline, 1, 2, 4 and 8 h) were performed in BEAS-2B cells alone, in BEAS-2B cells co-cultured with human neutrophils, in BEAS-2B cells co-co-cultured with human neutrophils and treated with bradykinin (10-6M) or LPS (1 μg/μl), in BEAS-2B cells co-cultured with human neutrophils and treated with bradykinin (10-6M) in combination with LPS (1 μg/μl). No data are reported after 8 h of incubation with human neutrophils because of a significant neutrophil diapedesis after this time point.
We also performed experiments on 3-NT formation by BEAS-2B cells and human neutrophils alone or in combination with bradykinin (10-6M) or LPS (1 μg/μl).
Finally, BEAS-2B cells co-cultured with human neutrophils were stimulated for 8 h with bradykinin (10-6M) and/or LPS (1 μg/μl) in order to assess 3-NT production by immunocytochemistry.
2.3. B2 receptor and 3-nitrotyrosine immunocytochemistry
Cytospin slides (Cytospin II; Shandon, London, UK) were prepared with pellets of BEAS-2B cells cultured in basal conditions and after a 12 h stimulation with bradykinin 10-6M for B2 receptor
expression or in basal condition and after 8 h stimulation with bradykinin 10-6M and/or LPS (1 μg/μl) in presence of human neutrophils for 3-NT production, respectively. B2 receptor expression and 3-NT formation were determined by immunocytochemistry on at least two cytospin for each condition (Emanuel et al., 2005).
For B2 receptor detection, slides were fixed in 4% p-formaldehyde (Sigma-Aldrich) for 20 minutes,
rehydrated, incubated in 3% hydrogen peroxide for 10 minutes at room temperature and, then, incubated over-night at 4°C with a mouse anti-human B2 receptor monoclonal antibody (BD
Biosciences, San Jose, CA, USA). 3,39-Diaminobenzidine (DAB) Substrate Chromogen System (Dako Cytomation, Carpinteria, CA, USA) was employed in the detection procedure.
For 3-NT detection, slides were fixed in 4% p-formaldehyde (Sigma-Aldrich) for 15 minutes, rehydrated and after blocking non-specific binding sites using serum derived from the same animal species as the secondary antibody, were incubated 1 h at room temperature in a humidified chamber with the rabbit anti-nitrotyrosine polyclonal primary antibody (1:300, Upstate Biotechnology, Lake Placid, NY, USA). Antibody binding was demonstrated with the use of a secondary anti rabbit antibody (Vector, BA 1000) followed by Strept AB Complex/AP (Dako, K0391) and fast-red substrate (Sigma-Aldrich).
To evaluate the cellular architecture, slides were stained with Harris’ haematoxylin solution (Merck, Darmstadt, Germany). The total number of cells and B2 receptor or 3-NT positive cells were
counted with a light microscope (BH-2; Olympus, Hamburg, Germany) in four or more randomly chosen high-power (magnification 400X) fields and the results were expressed as percentage of positive cells.
2.4. B2 receptor localization by immunofluorescence analysis
After 12 h incubation with bradykinin (10-6M), BEAS-2B cells were fixed in methanol for 5 minutes, treated with 10% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS) (Gibco) for 30 minutes (in order to remove aspecific signal), and incubated with mouse anti-human B2 receptor monoclonal antibody (diluted 1:100 in PBS), for 2 h at room temperature. Afterwards,
the antibody-antigen complexes were incubated for 30 minutes with a FITC-conjugated goat anti-mouse Alexa Fluor 488 secondary antibody (Molecular Probes, Invitrogen, Carlsbad, CA, USA) and labelled for 5 minutes with Propidium Iodide (Sigma-Aldrich) for nucleus staining. Cells were then analysed by a Confocal Scanning Microscope (LSM 510; Carl Zeiss MicroImaging, Inc.) (Oberkochen, Germany) and a 100X objective was used to capture images. Fluorescence was measured by Image J Software [National Institute of Health (NIH), Bethesda, USA]. Results were expressed as number of cells with co-localized fluorescence signals of B2 receptor and nucleic acids
co-localization of the two fluorescence signals (B2 receptor and nucleic acids) related to the number of
cells expressing the B2 receptor], for the expression of bradykinin B2 receptor (Peracino et al.,
1998).
2.5. Isolation of polymorphonuclear neutrophils
Polymorphonuclear neutrophils (PMNs) were isolated from peripheral blood of healthy volunteers by standard dextran sedimentation followed by Histopaque® (Sigma-Aldrich) gradient centrifugation. The PMNs were resuspended in Hepes Buffered Saline Solution (HBSS) (Clonetics, Basel, Switzerland). Final cell suspension purity averaged 98%. PMNs viability (assessed by the trypan blue exclusion test) was always greater than 95%. The study conformed to the declaration of Helsinki and informed consent was obtained from each volunteer.
2.6. Western blot analysis
To evaluate B2 receptor expression, BEAS-2B cells were incubated for 12 h with scalar
concentrations of bradykinin and at different time points with bradykinin 10-6M, as described above. After all treatments cells were washed in PBS and lysed in 1ml/dish of lysis buffer [20 mM Tris HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0,1% sodium dodecyl sulphate (SDS), 1mM ethylene glycol tetra acetic acid (EGTA), 1mM ethylene diamine tetra acetic acid (EDTA), 1mM Na ortho-vanadate and 1X Protease Inhibitor Cocktail (Sigma-Aldrich)]. Total cell lysates were incubated at 4°C for 1 h, centrifuged at 10.000 rpm for 10 minutes and their protein concentration quantified by the “Bio-Rad Protein Assay” (Bio-Rad Laboratories, Hercules, CA, USA). Protein extracts (70 μg/well) were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on nitrocellulose membranes (Bio-Rad). Blots were incubated with a mouse anti-B2 receptor antibody (BD Biosciences) and visualised using the
with anti-β-actin antibody (clone C4; Boehringer Mannheim Inc., Mannheim, Germany). The relevant band intensities were analysed by NIH Image J 1.38x program.
2.7. IL-8, MPO and 3-nitrotyrosine assays
BEAS-2B cells were incubated alone or in co-culture with human neutrophils after treatment with bradykinin, LPS, HOE 140 and Lys-(des-Arg9, Leu8)-bradykinin as previously described. IL-8 (PeproTech Ltd, London, UK), MPO (R&D Systems, Abingdon, UK) and 3-nitrotyrosine (Cell Biolabs Inc., San Diego, CA, USA) production was evaluated in the supernatants by enzyme-linked immunosorbent assays (ELISAs) following the manufacturer’s instructions. Results were expressed as ng/ml per 5x105 viable cells for IL-8 and MPO, and as μg/ml per 5x105 viable cells for 3-nitrotyrosine respectively.
Cell viability was evaluated by trypan blue exclusion test in each experimental set. Counting total number of cells was done by using a Burker counting chamber and a binocular microscope, at 200X magnification. Trypan blue exclusion (0.4% final dye concentration) was used for viability assessment. Estimations of the percentages of non-viable cells were made by counting the percentage of blue cells. Viability values were on average higher than 98%.
2.8. Statistical analysis
Statistical evaluation was performed using the statistical software package GraphPad Prism 3.0 (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± S.E.M. of three independent experiments (each experiment was performed in duplicate). Multiple groups were tested for significance by one-way ANOVA and post hoc Dunnett’s two-sided comparison test. For comparison between two groups an Unpaired t-test was performed. A P-value <0.05 was considered to be significant.
3.
Results
3.1. Immunostainings of B2 receptor expression in human bronchial epithelial cells BEAS-2B
By means of immunocytochemistry we showed that BEAS-2B cells rarely expressed B2 receptor
constitutively (Fig. 1A) and 12 h treatment with bradykinin (10-6M) significantly increased B2
receptor expression (23,4% ±0,21 of B2 receptor+ cells; Fig. 1C) compared to unstimulated
BEAS-2B cells at 12 h (3,5% ±0,30 of B2 receptor+ cells; P<0.001) (Fig. 1B). These data were confirmed
by confocal analysis (Fig. 1D and 1E), in which the enhanced B2 receptor expression on BEAS-2B
cells was evident after 12 h of stimulation with bradykinin (10-6M) (Fig. 1E and 1F).
3.2. Effect of bradykinin on B2 receptor protein expression in human bronchial epithelial cells
BEAS-2B
We performed a time course of bradykinin-induced B2 receptor protein expression in BEAS-2B
cells (bradykinin: 10-6M) showing a significant increased content of B2 receptor protein in response
to bradykinin at 8 and 12 h (P<0.05) (Fig. 2A).
We also performed a dose-response curve of bradykinin-induced B2 receptor protein expression in
BEAS-2B cells after 12 h of incubation assessed by Western blot analysis. As shown in figure 2B, BEAS-2B cells constitutively expressed B2 receptor and bradykinin significantly up-regulated
BEAS-2B cells expression of B2 receptor from 10-9M (P<0.05) to 10-5 M and 10-6M (P<0.001).
3.3. Effect of bradykinin and LPS on interleukin-8 secretion by human bronchial epithelial cells BEAS-2B
As shown in figure 3A, unstimulated BEAS-2B cells produced IL-8 release. In BEAS-2B cells IL-8 release in response to bradykinin (10-6M) increased at 8, 12 and 24 h of incubation (P<0.05), reaching the maximal increase at 12 and 24 h (Fig. 3A). We performed a dose-response curve of bradykinin-induced IL-8 release in BEAS-2B cells after 12 h of incubation (Fig. 3B). Bradykinin
significantly increased IL-8 secretion by BEAS-2B cells from 10-8M (P<0.05) to 10-5 M and 10-6 M (P<0.01). IL-8 release was also assayed in BEAS-2B cells incubated for 12 h after treatment with LPS (1 μg/μl) alone or in combination with bradykinin (10-6M) and with bradykinin alone (Fig. 3C). Bradykinin (P<0.05) or LPS alone (P<0.001) significantly enhanced IL-8 secretion in BEAS-2B; but the combination of the two stimuli did not provoke an additional increase in IL-8 release (P<0.001) (Fig. 3C).
Bradykinin-induced IL-8 secretion was partly inhibited by the pre-treatment with the B2 receptor
selective antagonist HOE 140 (1μM; 56,3% inhibition) or with the B1 receptor selective antagonist
Lys-(des-Arg9, Leu8)-bradykinin (1μM; 49,6 % inhibition) and almost suppressed after pre-treatment with both selective antagonists (89,5 % inhibition) as shown in figure 3D.
3.4. Effect of bradykinin and LPS on MPO release and 3-nitrotyrosine production by human bronchial epithelial cells BEAS-2B co-cultured with human neutrophils
MPO release and 3-nitrotyrosine production was evaluated at different time points (1, 2, 4, 8 h) in supernatants of BEAS-2B cells alone, of BEAS-2B cells co-cultured with human neutrophils, of BEAS-2B cells co-cultured with human neutrophils and treated with bradykinin (10-6M) or LPS (1 μg/μl) and of BEAS-2B cells co-cultured with human neutrophils treated with bradykinin (10-6
M) and LPS (1 μg/μl).
Levels of MPO measured in basal condition with BEAS-2B cells alone have been considered an unspecific signal of ELISA, therefore it has been subtracted from the values measured in the presence of human neutrophils that are the actual source of MPO.
The presence of human neutrophils in co-culture with BEAS-2B cells showed a significant time-dependent increase of MPO release. At all time points bradykinin or LPS induced a significant increase of MPO release from BEAS-2B cells in co-culture with human neutrophils compared to BEAS-2B cells in co-culture with human neutrophils alone (P<0.001), and the addition of both
bradykinin and LPS in BEAS-2B cells co-cultured with human neutrophils showed a further dramatical increase of MPO production (Fig. 4).
Levels of 3-NT measured in basal condition with BEAS-2B cells alone have been considered an unspecific signal of ELISA, therefore it has been subtracted from the values measured in the presence of human neutrophils. BEAS-2B cells in co-culture with human neutrophils at 2, 4 and 8 h increased 3-nitrotyrosine production in comparison with BEAS-2B cells with human neutrophils at 1 h (P<0.001; Fig. 5); in BEAS-2B cells co-cultured with human neutrophils, bradykinin significantly enhanced 3-nitrotyrosine production at 2, 4 and 8 h, reaching the maximum release at 8 h, compared to BEAS-2B cells in co-culture with human neutrophils (P<0.001). LPS was also able to increase 3-nitrotyrosine formation at 4 and 8 h compared to BEAS-2B in co-culture with human neutrophils (P<0.001); finally, the presence of both bradykinin and LPS in BEAS-2B cells co-cultured with human neutrophils provoked an additional increase of 3-nitrotyrosine production at 2, 4 and 8 h of incubation compared to the bradykinin or LPS alone (Fig. 5).
We point out that in co-cultures neutrophil diapedesis was not evident at initial time points (1, 2, 4 h) and at 8 h neutrophil diapedesis was less of 10% (data not shown). Furthermore, it was not possible to obtain data after 12 h of incubation with human neutrophils because of an elevated diapedesis.
3.5. Effect of bradykinin and LPS on 3-nitrotyrosine production by human bronchial epithelial cells BEAS-2B or human neutrophils
In BEAS-2B cells bradykinin (10-6M) or LPS (1 μg/μl) significantly enhanced 3-nitrotyrosine production at 1, 2, 4 and 8 h (P<0.01; Table 1). In particular, bradykinin was able to induce a time-dependent increase reaching the maximum production of 3-nitrotyrosine at 8 h compared to BEAS-2B cells alone (P<0.01; Table 1). Furthermore, we noted that LPS reached the maximum effect of 3-nitrotyrosine production in BEAS-2B cells at the first hour of incubation without variations at each time point (P<0.01; Table 1).
Human neutrophils alone are able to release the same amount of 3-nitrotyrosine at each time point (markedly higher than BEAS-2B cells alone; P<0.01). In presence of bradykinin (10-6M) human neutrophils significantly increased 3-nitrotyrosine formation maximally at 4 h (>2 fold; P<0.01) and, to a lesser extent, at 8 h (P<0.05), as shown in Table 1. In presence of LPS (1 μg/μl) human neutrophils significantly increased 3-nitrotyrosine formation at 4 h and at 8 h (>2 fold; P<0.01; Table 1).
3.6. Immunocytochemistry of 3-NT expression in human bronchial epithelial cells BEAS-2B
3-nitrotyrosine formation was evaluated at 8 h in BEAS-2B cells alone, in BEAS-2B cells co-cultured with human neutrophils, in BEAS-2B cells co-co-cultured with human neutrophils treated with bradykinin (10-6M) or LPS (1 μg/μl) and in BEAS-2B cells co-cultured with human neutrophils treated with bradykinin (10-6M) and LPS (1 μg/μl).
BEAS-2B cells rarely produced 3-NT constitutively (3,2 % ± 0,44 of 3-NT+ cells; Fig. 6A). The presence of human neutrophils in co-culture with BEAS-2B cells enhanced 3-NT formation compared to BEAS-2B alone (30 % ± 1,73 of 3-NT+ cells; Fig. 6B and 6F) and the treatment with bradykinin (10-6M) significantly increased 3-NT production (61,7 % ± 4,41 of 3-NT+ cells; Fig. 6C) compared to BEAS-2B cells co-cultured with human neutrophils (P<0.001) (Fig. 6F). In co-cultures of BEAS-2B cells and human neutrophils the treatment with LPS (1 μg/μl) also increased 3-NT formation (55 % ± 2,89 of 3-NT+ cells; Fig. 6D) compared to BEAS-2B cells co-cultured with human neutrophils (P<0.001) (Fig. 6F), but the highest production of 3-nitrotyrosine was obtained in BEAS-2B cells co-cultured with human neutrophils after the addition of both bradykinin (10-6M) and LPS (1 μg/μl) (92,3 % ± 3,38 of 3-NT+
cells; Fig. 6E) compared to BEAS-2B cells co-cultured with human neutrophils and treated with bradykinin or LPS (P<0.001) (Fig. 6F).
4.
Discussion
This study demonstrated that bradykinin is able to induce bradykinin B2 receptor protein expression
and that both bradykinin and LPS are able to increase IL-8 secretion in human bronchial epithelial cells BEAS-2B. In co-cultures of BEAS-2B cells with human neutrophils, we showed that both bradykinin and LPS are also able to induce MPO release and 3-nitrotyrosine production.
In our study we observed constitutive expression of B2 receptor and the capability of bradykinin, as
previously shown by Bengtson and co-workers (Bengtson et al., 2008), to induce an up-regulation of B2 receptor protein expression in BEAS-2B cells suggesting that kinins might activate
intracellular mechanisms, probably via transcription factors, able to generate protein de novo synthesis of B2 receptor (Brechter et al., 2008) and/or might regulate B2 receptor protein expression
at post-translational level (Simaan et al., 2005; Prado et al., 2002). In human airways, the effects of kinins are mostly mediated through B2 receptor (Abraham et al., 2006). B2 receptors are
constitutively expressed on many cell types including human airway epithelial cells (Abraham et al., 2006). Several G protein-couple receptors, including B2 receptor, have been shown to mediate
NF-kB activation and cytokine gene transcription in human epithelial cells and fibroblasts (Chen et al., 2004; Hayashi et al., 2000). Furthermore bradykinin, acting through B2 receptor, activates the
Ras/Raf-1/ERK which in turn initiates IKK and NF-kB activation in human airway epithelial cells (A549) (Chen et al., 2004) and also the p38 MAPK pathway in human lung fibroblasts (Hayashi et al., 2000). The intracellular pathways that regulate the expression of the B2 receptor
gene and those which are activated by B2 receptor stimulation in human cells are only partially
known (Haddad et al., 2000; Prado et al., 2002). In human osteoblastic cell line and gingival fibroblasts, TNF--induced B2 receptor gene expression involves NF-kB and p38 MAPK pathways
(Brechter et al., 2008).
We also demonstrated the capability of bradykinin and LPS to equally produce an enhanced secretion of IL-8 in bronchial epithelial cells without provoking an additional effect when both stimuli were combined. Moreover, the effect of bradykinin on IL-8 release in bronchial epithelial
cells was dependent on both B2 receptor and B1 receptor as showed by the inhibition of the selective
antagonists. We may also postulate that bradykinin provoked IL-8 release not only via activation of B2 receptor but also stimulating the B1 receptor after its degradation to [des-Arg9]-bradykinin
during the 12 h of incubation (Bathon et al., 1992). IL-8, a potent activator and chemoattractant of neutrophils (Rodgers et al., 2002), is increased in bronchoalveolar lavage fluid of several chronic respiratory diseases (Cetin et al., 2004; Riise et al., 1995) and in cultured airway epithelial cells by a number of stimuli, including cytokines (IL-1, TNF-, IFN-), Pseudomonas aeruginosa and respiratory viruses (Rodgers et al., 2002). In a previous study it has been demonstrated that bradykinin increased IL-8 release in airway epithelial cells through autocrine generation of endogenous prostanoids (Rodgers et al., 2002). Other studies in human lung fibroblasts showed that bradykinin produced IL-8 by increasing its gene expression (Hayashi et al., 1998) and also that bradykinin-induced IL-8 production, completely due to B2 receptor stimulation, is in part dependent
on the activation of ERK1/2 and p38 MAPK-pathway (Hayashi et al., 2000). Our study partly confirmed the former study because we found that bradykinin released IL-8 in bronchial epithelial cells via the stimulation of both B2 receptor and B1 receptor. Finally, our study is in line with
previous reports on the capability of LPS to provoke IL-8 production in bronchial epithelial cells without exploring the intimate mechanisms involved in LPS-induced IL-8 release: 1) Pseudomonas aeruginosa LPS stimulated the production of NF-kB-dependent IL-8 in primary cultures of bronchial epithelial cells from patients undergoing lung transplantation (Escotte et al., 2003); 2) in primary culture of small airway epithelial cells LPS caused IL-8 production via activation of protease-activated receptor 2 (PAR-2) (Ostrowska et al., 2007).
Myeloperoxidase (MPO), is the most abundant protein in neutrophils and represents 5% of their total protein content. MPO is produced as a precursor during myeloid differentiation in bone marrow, and its processing is completed before neutrophils enter the circulation. A hallmark of neutrophil activation is their respiratory burst, in which MPO plays a central role. MPO catalyses the conversion of hydrogen peroxide (H2O2) and chloride ions into hypochlorous acid (HOCl) and,
therefore, it is an important enzyme in the host defence against bacteria, viruses and fungi. MPO may also damage tissue by its production of HOCl and other reactive oxidants. HOCl production by MPO leads to the formation of chlorotyrosine residues, a marker of neutrophilic inflammation (Gujral et al., 2004). Nitric oxide (NO) and nitrite (NO2) also serve as biological substrates of MPO, leading to the formation of nitrating intermediates, in an inflammatory microenvironment featured by exaggerated production of NO in presence of oxidative stress (Ricciardolo, 2003; Ricciardolo et al., 2006; Sterk et al., 1999). Reactive nitrogen species (RNS), such as peroxynitrite (formed by the reaction between superoxide anion and NO) and nitrating intermediates, promote protein nitration classically detected by the reactive stable end-product 3-nitrotyrosine (Eiserich et al., 1998).
Numerous in vitro biochemical studies have demonstrated that nitration of protein tyrosine residues, per se, alter protein function, damage DNA, lipids and carbohydrates leading to impaired cellular functions and enhanced inflammatory reactions (Ricciardolo et al., 2006, 2004).
In our study, in co-cultures of bronchial epithelial cells BEAS-2B with human neutrophils we observed an increase of MPO release after stimulation with bradykinin (to a lesser extent) or LPS alone and in combination of both (exponentially higher) indicating that bradykinin is able per se to stimulate MPO release from activated neutrophils probably via IL-8-dependent bronchial epithelial pathway or also via direct activation of bradykinin receptors in neutrophils (Ehrenfeld et al., 2009) and that LPS is able to dramatically stimulate neutrophils with the subsequent MPO production either as a direct major stimulus via TLR-4 (Gomes et al., 2010) or an indirect stimulus via release of IL-8 by bronchial epithelial cells (Sha et al., 2004).
In our study we found that the production of 3-nitrotyrosine measured either in supernatants or in BEAS-2B cells was provoked either by bradykinin or LPS alone and in combination of both in the presence of human neutrophils suggesting that bradykinin has a specific role in the development of “nitrosative stress” in neutrophilic-dependent chronic airway inflammation and that LPS may contribute in the amplification of the inflammatory environment (Sha et al., 2004). Finally, we also show that both bradykinin and LPS stimulate BEAS-2B cells alone to generate “nitrosative stress”,
via a neutrophilic-independent pathway, probably through iNOS-induced NO release mediated by NF-kB activation (Chen et al., 2004, Jiang et al., 2000, Ricciardolo et al., 2004) in conjunction with oxidative stress produced by bronchial epithelial cells itself. In particular, we may speculate that the lower increase of bradykinin-induced 3-nitrotyrosine formation in BEAS-2B cells at early time points was probably due to the release of low amount of NO by the activation of cNOS after intracellular calcium mobilization following bradykinin B2R stimulation (Ricciardolo et al., 2000).
5.
Conclusions
In conclusion, the current study showed that bradykinin was able to increase the protein expression of the constitutive B2 receptor and that both bradykinin and LPS, two pro-inflammatory stimuli,
were able to release the neutrophil chemoattractant IL-8 in BEAS-2B cells. The novelty of our study is the proof of evidence concerning the capability of bradykinin and LPS to enhance the production of MPO and 3-nitrotyrosine, demonstrating the formation of “nitrosative stress”, in a model of bronchial epithelial cells cultured alone or with human neutrophils. Because a long-term persistence of “nitrosative stress” might contribute to the progressive deterioration of pulmonary functions leading to respiratory failure, further studies should be conducted to better investigate the bradykinin- and LPS-mediated pathways involved in “nitrosative stress”, in order to hypothesize a potential modulation of this process by targeting bradykinin and/or LPS receptors and/or the subsequent signalling pathways, possibly leading to new therapeutic horizons.
Acknowledgments
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Figure captions
Figure 1. B2 receptor expression in BEAS-2B cells. B2 receptor immunocytochemistry (Upper
panels): BEAS-2B cells were cultured in basal conditions (A; 0% positive cells) and for 12 h in absence (B; 3% positive cells) or presence (C; 25% positive cells) of bradykinin (10-6M). The results were expressed as percentage of B2 receptor positive cells (Original magnification 400X). B2
receptor immunofluorescence (Lower panels): BEAS-2B cells were cultured in basal conditions (D) and for 12 h in presence of bradykinin (10-6M) (E). Fluorescence was quantified by color intensity (F). Red: Propidium Iodide; Green: B2 receptor; MGV: mean gray value. Each bar indicates the
mean value in the respective group. **: P<0.01.
Figure 2. Effect of bradykinin on B2 receptor production by BEAS-2B cells. Cells were incubated with bradykinin (10-6M) for the time indicated (A), a dose-response curve was performed after 12 h of incubation with bradykinin (B) and B2 receptor expression was determined by Western blot
analysis. Data are presented as mean ± S.E.M. from three independent experiments. BK=bradykinin; B2R=B2 receptor. *: P<0.05; **: P<0.01; ***: P<0.001.
Figure 3 Effect of bradykinin on interleukin (IL)-8 secretion by BEAS-2B cells. Cells were
incubated with bradykinin (10-6M) for the time indicated (A) and a dose-response curve was performed after 12 h of incubation with bradykinin (B). Cells were incubated with LPS (1 g/l) for 12 h both in absence or presence of bradykinin (10-6M) (C). Bradykinin-induced IL-8 release (at 12 h) after pre-treatment with the B2 receptor selective antagonist HOE 140 (1μM; for 1 h) or with
the B1 receptor selective antagonist Lys-(des-Arg9, Leu8)-bradykinin (1μM; for 1 h) and with both
the selective antagonists in BEAS-2B cells (D). IL-8 secretion was determined by enzyme-linked immunosorbent assay in the supernatants. The results are expressed as mean ± S.E.M. of three
independent experiments. BK=bradykinin; B1R Ant= B1 receptor selective antagonist
Lys-(des-Arg9, Leu8)-bradykinin. *: P<0.05; **: P<0.01; ***: P<0.001.
Figure 4. Effect of bradykinin (10-6M) or LPS (1 μg/μl) on MPO release in BEAS-2B cells
co-cultured with human neutrophils and of both stimuli in BEAS-2B cells co-co-cultured with human neutrophils at different time points (1, 2, 4, 8 h). MPO was determined by enzyme-linked immunosorbent assay in the supernatants. The results are expressed as mean ± S.E.M. of three independent experiments. BK=bradykinin; HN=Human Neutrophils. ***: P<0.001.
Figure 5. Effect of bradykinin (10-6M) or LPS (1 μg/μl) on 3-nitrotyrosine production in BEAS-2B
cells co-cultured with human neutrophils and of both stimuli in BEAS-2B cells co-cultured with human neutrophils at different time points (1, 2, 4, 8 h). 3-nitrotyrosine was determined by enzyme-linked immunosorbent assay in the supernatants. The results are expressed as mean ± S.E.M. of three independent experiments. BK=bradykinin; HN=Human Neutrophils. ***: P<0.001.
Figure 6. 3-nitrotyrosine (3-NT) production in BEAS-2B cells. 3-NT immunocytochemistry: BEAS-2B cells were cultured in basal condition (panel A; 3% positive cells) and co-cultured with human neutrophils alone (panel B; 30% positive cells), in presence of bradykinin (10-6M) (panel C; 62% positive cells), LPS (1 μg/μl) (panel D; 55% positive cells) or the combination of both stimuli (panel E; 92% positive cells) for 8 h. Original magnification 400X. The results were expressed as percentage of 3-NT positive cells (panel F). Data are presented as mean ± S.E.M. from three independent experiments. BK=bradykinin; HN=Human Neutrophils. **: P<0.01; ***: P<0.001.
