Effect of Ambroxol and Beclomethasone on Lipopolysaccharide-Induced Nitrosative Stress in Bronchial Epithelial Cells.

BACKGROUND: Nitrosative stress is involved in different airway diseases. Lipopolysaccharide (LPS) induces neutrophil-related cytokine release and nitrosative stress in human bronchial epithelial (BEAS-2B) cells alone or with human polymorphonuclear neutrophils (PMNs). Ambroxol protects against oxidative stress, and beclomethasone dipropionate is an anti-inflammatory drug. OBJECTIVES: We evaluated the ability of ambroxol and/or beclomethasone dipropionate to inhibit LPS-induced expression/release of RANTES, IL-8, inducible NO synthase (iNOS), myeloperoxidase (MPO) and 3-nitrotyrosine (3-NT: nitrosative stress biomarker) in BEAS-2B ± PMNs stimulated with LPS (1 µg/ml). METHODS: The effect of ambroxol and/or beclomethasone dipropionate on IL-8, RANTES and iNOS levels was assessed by Western blot analysis; IL-8, MPO and 3-NT levels were measured by ELISA. Cell viability was assessed by the trypan blue exclusion test. RESULTS: In BEAS-2B alone, LPS (at 12 h) increased RANTES/iNOS expression and IL-8 levels (p < 0.001). Ambroxol suppressed LPS-induced RANTES expression and IL-8 release (p < 0.001), whilst inhibiting iNOS expression (p < 0.05). Beclomethasone dipropionate had no effect on RANTES but halved iNOS expression and IL-8 release. Coculture of BEAS-2B with PMNs stimulated IL-8, MPO and 3-NT production (p < 0.001), potentiated by LPS (p < 0.001). Ambroxol and beclomethasone dipropionate inhibited LPS-stimulated IL-8, MPO and 3-NT release (p < 0.05). Ambroxol/beclomethasone dipropionate combination potentiated the inhibition of IL-8 and 3-NT production in BEAS-2B with PMNs (p < 0.05 and p < 0.01, respectively). Ambroxol and/or beclomethasone dipropionate inhibited nitrosative stress and the release of neutrophilic inflammatory products in vitro. CONCLUSION: The additive effect of ambroxol and beclomethasone dipropionate on IL-8 and 3-NT inhibition suggests new therapeutic options in the treatment of neutrophil-related respiratory diseases such as chronic obstructive pulmonary disease and respiratory infections.

Expression of vascular remodelling markers in relation to bradykinin receptors in asthma and COPD.

BACKGROUND: Vascular remodelling plays a central role in asthma and chronic obstructive pulmonary disease (COPD). Bradykinin (BK) is a vasoactive proinflammatory peptide mediating acute responses in asthma. We investigated the role of angiogenic factors in relation to BK receptors in asthma and COPD. METHODS: Bronchial biopsies from 33 patients with COPD, 24 old (≥50 years) patients with (≥50 years) asthma, 18 old control smokers, 11 old control non-smokers, 15 young (≤40yrs) patients with (≤40 years) asthma and 10 young control non-smokers were immunostained for CD31, vascular endothelial growth factor-A (VEGF-A), angiogenin and BK receptors (B2R and B1R). Fibroblast and endothelial co-localisation of relevant molecules were performed by immunofluorescence. BK-induced VEGF-A and angiogenin release was studied (ELISA) in bronchial fibroblasts from subjects with asthma and COPD. RESULTS: In bronchial lamina propria of old patients with asthma, CD31 and VEGF-A(+) cell numbers were higher than old control non-smokers (p<0.05). Angiogenin(+), B2R(+) and B1R(+) cell numbers in old patients with asthma were higher than in old control non-smokers, control smokers and patients with COPD (p<0.01). Angiogenin(+) cell numbers were higher in patients with COPD than both old control groups (p<0.05). In all patients with asthma the number of B2R(+) cells was positively related to the numbers of B1R(+) (rs=0.43), angiogenin(+) (rs=0.42) and CD31 cells (rs=0.46) (p<0.01). Angiogenin(+) cell numbers were negatively related to forced expiratory volume in 1 s (rs=-0.415, p=0.008). Double immunofluorescence revealed that CD31 cells of capillary vessels coexpressed B2R and that fibroblasts coexpressed B2R, VEGF-A and angiogenin. BK (10(-6)M) induced significant angiogenin release in fibroblasts from asthma and to a lesser extent in COPD. CONCLUSIONS: Unlike COPD, this study suggests the involvement of BK receptors in bronchial vascular remodelling in asthma.

Bradykinin- and lipopolysaccharide-induced bradykinin B2 receptor expression, interleukin 8 release and "nitrosative stress" in bronchial epithelial cells BEAS-2B: role for neutrophils.

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 B(2) 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 B(2) 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 B(2) receptor expression (P<0.05) and stimulate IL-8 release (P<0.001) in BEAS-2B cells. Either the selective bradykinin B(2) receptor antagonist HOE 140 or the selective bradykinin B(1) receptor antagonist Lys-(des-Arg(9), Leu(8))-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".

Magnetically enriched bone marrow-derived macrophages loaded in vitro with iron oxide can migrate to inflammation sites in mice.

In vitro labelling of cells permits incorporation of large amounts of iron oxide and consequently high detection sensitivity, but it remains controversial whether labelled cells would respond normally to stimuli. This question was addressed by differentiating bone marrow-derived macrophages (BMDMs) in vitro, labelling cells with high concentrations of Endorem in vitro, and eliminating unlabelled cells by magnetic enrichment. To explore their acute inflammatory response, enriched cells were injected into mice with carrageenan-induced inflammation, the 'air pouch model'. Cells recovered from the inflammation site 16 h after intravenous BMDM injection into the tail vein were analysed by in vitro MRI and fluorescent microscopy. With both assays, Endorem-labelled cells were detectable. This indicates that BMDMs, loaded with high concentrations of iron oxide in vitro, can still respond to chemokine gradients and infiltrate inflamed tissue in mice. Furthermore, by using genetically modified mice as BMDM donors, it should be possible to study the role of individual genes in macrophage recruitment.

Quantification of the expression level of integrin receptor alpha(v)beta3 in cell lines and MR imaging with antibody-coated iron oxide particles.

Targeted imaging requires site-specific accumulation of a contrast agent (CA), and the properties of that agent must be selected according to the abundance of the target to obtain a signal above the detection limit of the instrument. However, numerical estimates of receptors per cell are rarely found in the literature. Integrin receptors would be particularly promising targets because of their accessibility from the blood stream and expression on activated neovascular endothelial cells. We systematically estimated the number of integrin receptors of cell lines and primary cells by flow cytometry analysis. Since integrin receptors are heterodimeric molecules, and alpha(v) forms complexes with various beta subunits, the numbers of alpha(v) and beta(3) subunits are therefore dissimilar. The observed values are 3 . 10(3)-1.4 . 10(4)/cell for alpha(v), and 5.3 . 10(2)-1.1 . 10(4)/cell for beta(3). Despite the low number of exposed receptors, we show that up to single-cell MR visualization can be achieved with the use of iron oxide beads complexed with antibodies as CAs.