Immunobiology 219 (2014) 392–401
Contents lists available at ScienceDirect
Immunobiology journal homepage: www.elsevier.com/locate/imbio
Increased levels of Th17 cells are associated with non-neuronal acetylcholine in COPD patients Mirella Profita a,∗ , Giusy Daniela Albano a,b , Loredana Riccobono a , Caterina Di Sano a , Angela Marina Montalbano a , Rosalia Gagliardo a , Giulia Anzalone a , Anna Bonanno a , Michael Paul Pieper c , Mark Gjomarkaj a a Unit: “Ex vivo/In vitro Models to Study the Immunopathology and the Pharmacology of Airway Diseases”, Institute of Biomedicine and Molecular Immunology (IBIM), Italian National Research Council (CNR), Palermo, Italy b Dipartimento Biomedico di Medicina, Interna e Specialistica (Di.Bi.M.I.S.), Sezione di Pneumologia, University of Palermo, Palermo, Italy c Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany
a r t i c l e
i n f o
Article history: Received 29 January 2013 Received in revised form 19 July 2013 Accepted 9 January 2014 Available online 25 January 2014 Keywords: Th-17 cells Systemic inflammation Anticholinergic drugs Beta-2 long acting
a b s t r a c t T-lymphocytes, including Th17-cells and T-cells expressing acetylcholine (ACh), are key components of systemic inflammation in chronic obstructive pulmonary disease (COPD). We investigated whether ACh promotes Th17 cells in COPD. ACh, IL-17A, IL-22, ROR␥t, FOXP3 expression and AChIL-17A, AChIL-22, AChROR␥t coexpression was evaluated in peripheral blood mononuclear cells (PBMC) from COPD patients (n = 16), healthy smokers (HS) (n = 12) and healthy control subjects (HC) (n = 13) (cultured for 48 h with PMA) by flow cytometry. Furthermore, we studied the effect of Tiotropium (Spiriva® ) (100 nM) and Olodaterol (1 nM) alone or in combination, and of hemicholinium-3 (50 M) on AChIL-17A, AChIL-22, AChROR␥t, and FOXP3 expression in CD3+ PBT-cells of PBMC from COPD patients (n = 6) cultured for 48 h with PMA. CD3+ PBT-cells expressing ACh, IL-17A, IL-22 and ROR␥t together with CD3+ PBT-cells co-expressing AChIL-17A, AChIL-22 and AChROR␥t were significantly increased in COPD patients compared to HC and HS subjects with higher levels in HS than in HC without a significant difference. CD3+ FOXP3+ PBT-cells were increased in HS than in HC and COPD. Tiotropium and Olodaterol reduced the percentage of CD3+ PBTcells co-expressing AChIL-17A, AChIL-22, and AChROR␥t while increased the CD3+ FOXP3+ PBT-cells in PBMC from COPD patients, cultured in vitro for 48 h, with an additive effect when used in combination. Hemicholnium-3 reduced the percentage of ACh+ IL-17A+ , ACh+ IL-22+ , and ACh+ ROR␥t+ while it did not affect FOXP3+ expression in CD3+ PBT-cells from cultured PBMC from COPD patients. We concluded that ACh might promote the increased levels of Th17-cells in systemic inflammation of COPD. Long-acting 2-agonists and anticholinergic drugs might contribute to control this event. © 2014 Elsevier GmbH. All rights reserved.
Background Adaptive immune processes are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). It has been hypothesized that susceptibility to COPD may arise by a shift from the non-specific innate response present in every smoker toward
Abbreviations: ACh, acetylcholine; COPD, chronic obstructive pulmonary disease; CSE, cigarette smoke extract; FOXP3, Forkhead family transcription factor; HCh, hemicholinium; IL-17, Interleukin-17; mAChRs, muscarinic receptors; nAChRs, nicotinic receptors; PBMC, peripheral blood mononuclear cells; ROR␥t, retinoic acidrelated orphan receptor ␥t; Th, T helper. ∗ Corresponding author at: Institute of Biomedicine and Molecular Immunology, Italian National Research Council (CNR), Via Ugo La Malfa, 153, 90146 Palermo, Italy. Tel.: +39 0916809121; fax: +39 0916809122. E-mail address: profi[email protected] (M. Profita). 0171-2985/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.imbio.2014.01.004
an adaptive immune response with features typical of autoimmune processes (Cosio et al. 2002; Lee et al. 2007). Th17-cells have been recently described effector T-cell subsets characterized by the production of IL-17A, IL-17F, and IL-22, which have been implicated in the pathogenesis of several inflammatory and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and psoriasis (Ikeuchi et al. 2005; Wolk et al. 2009). IL-17A expression has also been detected in the target tissue during the progression of diseases (Steinman 2007). Furthermore, IL-17A is a pro-inflammatory cytokine that regulates recruitment of inflammatory cells including neutrophils and lymphocytes into the inflamed tissues by secretion of chemokines CXCL8 and CCL20 (Jones and Chan 2002; Kao et al. 2005). IL-17A can be produced by CD4 and CD8 T-cells of both type 1 (Th1) and type 2 (Th2) cytokine profiles that differentiate in Th17 by the activation of the nuclear receptor retinoic acid-related
M. Profita et al. / Immunobiology 219 (2014) 392–401
orphan receptor ␥t (ROR␥t) (Kao et al. 2005). A balance between Th17 and regulatory T-cells (Tregs) is crucial for immune homeostasis (Bettelli et al. 2006). An excess in Th17 function, or increased numbers of Th17 cells, and a defect in Treg function or reduced numbers of Tregs, may trigger the development and progression of inflammatory diseases, including allergic asthma and rhinitis and COPD. Tregs dominantly express Forkhead family transcription factor FOXP3 which activates many suppressive genes in Tregs and inhibits many effector T-cell genes. Pharmacological treatment might restore the balance between Th17 and Treg promoting the resolution of systemic and local inflammation in airway diseases (Eiastein and Williams 2009). Th17-cells produce IL-22, which has been linked to chronic inflammatory diseases (Ikeuchi et al. 2005; Wolk et al. 2009) with an important role in host defense against extracellular bacterial infections in the lung (Aujla et al. 2008). IL-22 positive cells are increased in the bronchial submucosa and in the bronchial epithelial cells from COPD patients (Di Stefano et al. 2009) and the pathological role of IL-22 is seen only in the presence of IL-17A, since it is involved in airway inflammation acting in synergy with IL-17A (Sonnenberg et al. 2010). However, although COPD might have features typical of autoimmune processes there are little data on the role IL-17A, IL-22 and Th17-cells in the peripheral blood of this disease. T-lymphocytes are one of the key components of inflammation in COPD (O’Donnell et al. 2006) and possess all the components required to constitute an independent, non-neuronal cholinergic system. These include acetylcholine (ACh), choline acetyltransferase (ChAT), its synthesizing enzyme, and muscarinic ACh receptors (mAChRs) (Profita et al. 2012a). These components can act as immunomodulator and modify T and B cell function via mAChR mediated pathways (Kawashima and Fujii 2008; Fujii et al. 2008). M3 AChR subtype and ChAT expression and the CD3+ and CD8+ ACh-binding are increased in PBT-cells from COPD patients (Profita et al. 2012b). Furthermore, in murine spleen T-cells the mAChRs stimulation enhances IL-10 and IL-17A and inhibits INF␥ secretion suggesting a plasticity of the T-cell cholinergic system (Qian et al. 2011). The first aim of this study was to investigate whether, in PBT from COPD patients “in vitro”, there is a relationship between the components of non-neuronal cholinergic system and Th17 cells. Furthermore to promote novel therapeutic strategies aimed to the resolution of systemic inflammation in COPD, we tested whether the anticholinergic drugs together with a long acting 2-agonists normally used in the treatment of these patients might be able to control the Th17 cells.
393
All patients were characterized with respect to gender, age, smoking history, COPD symptoms, co-morbidities and current treatment. Patients with a history of allergy, allergic rhinitis or asthma were excluded. Exclusion criteria included the following: active infection, current or previous diagnoses of rheumatological, malignant or other systemic inflammatory diseases, respiratory tract infections and treatment with glucocorticoids, 2-agonists or anticholinergics within 1-month prior the study.
Pulmonary function tests Pulmonary function tests included measurements of FEV1 and FVC and PEF. To assess the reversibility of the airway obstruction in subjects with a baseline FEV1% less than 80% of predicted. Data are expressed as percentage of predicted values. The FEV1 measurement was repeated 15 min after the inhalation of 200 g of salbutamol.
Isolation of human peripheral blood mononuclear cells (PBMC) Blood samples were collected in EDTA vacutainer tubes (BD Biosciences) and used for plasma selection and PBMC isolation from HC, HS and COPD patients. The cells were isolated using a density gradient centrifugation (Ficoll-paqueTM PLUS; Amersham Biosciences SE-751 84, Uppsala, Sweden) and, after two washes, the cells were suspended in RPMI 1640 cell culture medium (Invitrogen Life Technologies, Italy) supplemented with 10% heatinactivated fetal bovine serum (FBS), 2 mM l-glutamine, 20 mM HEPES, 100 U/ml penicillin, 100 g/ml streptomycin, 5 × 10−5 M 2-ME and 85 g/ml gentamicin. Purity and viability of the cells was tested using trypan blue exclusion. PBMC (1 × 106 cells/ml) recovered from HC, HS and COPD were cultured in the presence of PMA (50 ng/ml final concentration) (Sigma Aldrich) and ionomycin calcium salt (250 ng/ml final concentration) (Sigma Aldrich) for 48 h in 24-well cell culture plates in complete medium RPMI 1640 (Invitrogen Life Technologies, Italy) plus 10% heat-inactivated FBS. Cultured PBMC were assessed for intracellular cytokines, signal transduction and ACh by flow cytometry as described below.
Stimulation of human PBMC from COPD patients with drugs Materials and methods Patients All subjects were patients from the Section of Respiratory Medicine of the Italian National Research Council, Palermo, Italy. The local Ethical Committee approved the study, and participating subjects gave their written informed consent. The study was carried out using samples of three groups of subjects: 13 healthy asymptomatic non-smoking subjects with normal lung function (HC); 12 asymptomatic smokers with normal lung function (HS); 16 patients with chronic obstructive pulmonary disease (COPD). The diagnosis of COPD and the assessment of its severity were defined and classified according to the criteria reported by the Global Initiative for Obstructive Lung Disease (GOLD) guidelines for COPD management (GOLD stage ≥I) (Pauwels et al. 2001). COPD subjects with exacerbations within 1-month prior to the study were excluded. Patients with COPD and HS had a smoking history of 10 pack years or more.
PBMC (1 × 106 cells/ml) from COPD patients (n = 6) were cultured for 48 h in 24-well cell culture plates as previously described, in the presence or absence of the anticholinergic, Tiotropium Spiriva® (100 nM) (Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany) and the 2-AR-agonist, Olodaterol (1 nM) (Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany), alone or in combination. Furthermore, we studied the effect of hemicholinium (HCh-3) (50 M, Sigma–Aldrich), a potent and selective choline uptake blocker, in PBMC from COPD patients cultured for 48 h and compared with the anticholinergic compound Tiotropium (100 nM). The cell viability was evaluated by trypan blue exclusion at the end of each experiments, to exclude the toxicity of the drugs. After PBMC stimulation with drugs, the cells were harvested and stained with fluorescent-labeled antibodies for surface staining, intracellular cytokine, signal transduction and ACh expression and analyzed by flow cytometry as described afterwards.
394
M. Profita et al. / Immunobiology 219 (2014) 392–401
Fig. 1. Gating strategy for flow cytometric identification of lymphocyte subpopulations. The total lymphocytes were gated by forward and side scatter (red colored R1). The lymphocyte gate is further analyzed for its expression of CD3, CD4, CD8 and CD4/CD25 and each positive subpopulations were gated as R2 to allow respectively ulterior targeted analysis.
Stimulation of human PBMC from HC with ACh and CSE PBMC from HC (n = 6) and from COPD (n = 6) were cultured for 48 h in 24-well cell culture plates as previously described were cultured in the presence or absence of ACh (10 M) for 48 h. CSE was prepared as previously described (Profita et al. 2008) and further diluted to the required concentration of 5% in fresh culture medium. PBMC from HC (n = 6) cultured in the same conditions, were treated with and without cigarette smoke extract (CSE) 5% for 24 h, in the presence or absence of Tiotropium (100 nM). The viability of the cells exposed to CSE and Tiotropium was evaluated by trypan blue exclusion dye assay.
After stimulation, the cells were harvested and stained with fluorescent-labeled antibodies for surface staining, intracellular cytokine, signal transduction and ACh expression and analyzed by flow cytometry as described afterwards. Cell surface staining of human PBMC To describe the Peripheral Blood T-cell (PBT) phenotypes in PBMC from HC and HS subjects and from COPD patients, the following fluorochrome conjugated antibodies were used in different combinations: anti-CD3 (HIT3a, BD Pharmingen), anti-CD8 (BW135/80, Miltenyi Biotec Inc, Germany), anti-CD8 (RPA-T8, BD Pharmingen), anti-CD4 (M-T466, Miltenyi Biotec Inc., Germany),
Table 1 Demographic characteristics of patients.
Age (years) Sex, n (Male/Female) FEV1 (% predicted) FVC (% predicted) FEV1/FVC ratio% Smoking pack/yr
HC
HS
COPD
p value
(n = 13)
(n = 12)
(n = 16)
HC vs. HS
HC vs. COPD
HS vs. COPD
60 ± 7 6/7 125.5 ± 26.8 106.6 ± 15.3 88.5 ± 6.4 0
64 ± 3 7/5 105.5 ± 5.0 95.5 ± 3.5 95.0 ± 3.5 45.7 ± 18.3
66 ± 14 8/8 63.7 ± 10.2 70.6 ± 11.2 63.4 ± 7.0 42.0 ± 10.2
ns – ns ns ns O.001
ns –
Contents lists available at ScienceDirect
Immunobiology journal homepage: www.elsevier.com/locate/imbio
Increased levels of Th17 cells are associated with non-neuronal acetylcholine in COPD patients Mirella Profita a,∗ , Giusy Daniela Albano a,b , Loredana Riccobono a , Caterina Di Sano a , Angela Marina Montalbano a , Rosalia Gagliardo a , Giulia Anzalone a , Anna Bonanno a , Michael Paul Pieper c , Mark Gjomarkaj a a Unit: “Ex vivo/In vitro Models to Study the Immunopathology and the Pharmacology of Airway Diseases”, Institute of Biomedicine and Molecular Immunology (IBIM), Italian National Research Council (CNR), Palermo, Italy b Dipartimento Biomedico di Medicina, Interna e Specialistica (Di.Bi.M.I.S.), Sezione di Pneumologia, University of Palermo, Palermo, Italy c Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany
a r t i c l e
i n f o
Article history: Received 29 January 2013 Received in revised form 19 July 2013 Accepted 9 January 2014 Available online 25 January 2014 Keywords: Th-17 cells Systemic inflammation Anticholinergic drugs Beta-2 long acting
a b s t r a c t T-lymphocytes, including Th17-cells and T-cells expressing acetylcholine (ACh), are key components of systemic inflammation in chronic obstructive pulmonary disease (COPD). We investigated whether ACh promotes Th17 cells in COPD. ACh, IL-17A, IL-22, ROR␥t, FOXP3 expression and AChIL-17A, AChIL-22, AChROR␥t coexpression was evaluated in peripheral blood mononuclear cells (PBMC) from COPD patients (n = 16), healthy smokers (HS) (n = 12) and healthy control subjects (HC) (n = 13) (cultured for 48 h with PMA) by flow cytometry. Furthermore, we studied the effect of Tiotropium (Spiriva® ) (100 nM) and Olodaterol (1 nM) alone or in combination, and of hemicholinium-3 (50 M) on AChIL-17A, AChIL-22, AChROR␥t, and FOXP3 expression in CD3+ PBT-cells of PBMC from COPD patients (n = 6) cultured for 48 h with PMA. CD3+ PBT-cells expressing ACh, IL-17A, IL-22 and ROR␥t together with CD3+ PBT-cells co-expressing AChIL-17A, AChIL-22 and AChROR␥t were significantly increased in COPD patients compared to HC and HS subjects with higher levels in HS than in HC without a significant difference. CD3+ FOXP3+ PBT-cells were increased in HS than in HC and COPD. Tiotropium and Olodaterol reduced the percentage of CD3+ PBTcells co-expressing AChIL-17A, AChIL-22, and AChROR␥t while increased the CD3+ FOXP3+ PBT-cells in PBMC from COPD patients, cultured in vitro for 48 h, with an additive effect when used in combination. Hemicholnium-3 reduced the percentage of ACh+ IL-17A+ , ACh+ IL-22+ , and ACh+ ROR␥t+ while it did not affect FOXP3+ expression in CD3+ PBT-cells from cultured PBMC from COPD patients. We concluded that ACh might promote the increased levels of Th17-cells in systemic inflammation of COPD. Long-acting 2-agonists and anticholinergic drugs might contribute to control this event. © 2014 Elsevier GmbH. All rights reserved.
Background Adaptive immune processes are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). It has been hypothesized that susceptibility to COPD may arise by a shift from the non-specific innate response present in every smoker toward
Abbreviations: ACh, acetylcholine; COPD, chronic obstructive pulmonary disease; CSE, cigarette smoke extract; FOXP3, Forkhead family transcription factor; HCh, hemicholinium; IL-17, Interleukin-17; mAChRs, muscarinic receptors; nAChRs, nicotinic receptors; PBMC, peripheral blood mononuclear cells; ROR␥t, retinoic acidrelated orphan receptor ␥t; Th, T helper. ∗ Corresponding author at: Institute of Biomedicine and Molecular Immunology, Italian National Research Council (CNR), Via Ugo La Malfa, 153, 90146 Palermo, Italy. Tel.: +39 0916809121; fax: +39 0916809122. E-mail address: profi[email protected] (M. Profita). 0171-2985/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.imbio.2014.01.004
an adaptive immune response with features typical of autoimmune processes (Cosio et al. 2002; Lee et al. 2007). Th17-cells have been recently described effector T-cell subsets characterized by the production of IL-17A, IL-17F, and IL-22, which have been implicated in the pathogenesis of several inflammatory and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and psoriasis (Ikeuchi et al. 2005; Wolk et al. 2009). IL-17A expression has also been detected in the target tissue during the progression of diseases (Steinman 2007). Furthermore, IL-17A is a pro-inflammatory cytokine that regulates recruitment of inflammatory cells including neutrophils and lymphocytes into the inflamed tissues by secretion of chemokines CXCL8 and CCL20 (Jones and Chan 2002; Kao et al. 2005). IL-17A can be produced by CD4 and CD8 T-cells of both type 1 (Th1) and type 2 (Th2) cytokine profiles that differentiate in Th17 by the activation of the nuclear receptor retinoic acid-related
M. Profita et al. / Immunobiology 219 (2014) 392–401
orphan receptor ␥t (ROR␥t) (Kao et al. 2005). A balance between Th17 and regulatory T-cells (Tregs) is crucial for immune homeostasis (Bettelli et al. 2006). An excess in Th17 function, or increased numbers of Th17 cells, and a defect in Treg function or reduced numbers of Tregs, may trigger the development and progression of inflammatory diseases, including allergic asthma and rhinitis and COPD. Tregs dominantly express Forkhead family transcription factor FOXP3 which activates many suppressive genes in Tregs and inhibits many effector T-cell genes. Pharmacological treatment might restore the balance between Th17 and Treg promoting the resolution of systemic and local inflammation in airway diseases (Eiastein and Williams 2009). Th17-cells produce IL-22, which has been linked to chronic inflammatory diseases (Ikeuchi et al. 2005; Wolk et al. 2009) with an important role in host defense against extracellular bacterial infections in the lung (Aujla et al. 2008). IL-22 positive cells are increased in the bronchial submucosa and in the bronchial epithelial cells from COPD patients (Di Stefano et al. 2009) and the pathological role of IL-22 is seen only in the presence of IL-17A, since it is involved in airway inflammation acting in synergy with IL-17A (Sonnenberg et al. 2010). However, although COPD might have features typical of autoimmune processes there are little data on the role IL-17A, IL-22 and Th17-cells in the peripheral blood of this disease. T-lymphocytes are one of the key components of inflammation in COPD (O’Donnell et al. 2006) and possess all the components required to constitute an independent, non-neuronal cholinergic system. These include acetylcholine (ACh), choline acetyltransferase (ChAT), its synthesizing enzyme, and muscarinic ACh receptors (mAChRs) (Profita et al. 2012a). These components can act as immunomodulator and modify T and B cell function via mAChR mediated pathways (Kawashima and Fujii 2008; Fujii et al. 2008). M3 AChR subtype and ChAT expression and the CD3+ and CD8+ ACh-binding are increased in PBT-cells from COPD patients (Profita et al. 2012b). Furthermore, in murine spleen T-cells the mAChRs stimulation enhances IL-10 and IL-17A and inhibits INF␥ secretion suggesting a plasticity of the T-cell cholinergic system (Qian et al. 2011). The first aim of this study was to investigate whether, in PBT from COPD patients “in vitro”, there is a relationship between the components of non-neuronal cholinergic system and Th17 cells. Furthermore to promote novel therapeutic strategies aimed to the resolution of systemic inflammation in COPD, we tested whether the anticholinergic drugs together with a long acting 2-agonists normally used in the treatment of these patients might be able to control the Th17 cells.
393
All patients were characterized with respect to gender, age, smoking history, COPD symptoms, co-morbidities and current treatment. Patients with a history of allergy, allergic rhinitis or asthma were excluded. Exclusion criteria included the following: active infection, current or previous diagnoses of rheumatological, malignant or other systemic inflammatory diseases, respiratory tract infections and treatment with glucocorticoids, 2-agonists or anticholinergics within 1-month prior the study.
Pulmonary function tests Pulmonary function tests included measurements of FEV1 and FVC and PEF. To assess the reversibility of the airway obstruction in subjects with a baseline FEV1% less than 80% of predicted. Data are expressed as percentage of predicted values. The FEV1 measurement was repeated 15 min after the inhalation of 200 g of salbutamol.
Isolation of human peripheral blood mononuclear cells (PBMC) Blood samples were collected in EDTA vacutainer tubes (BD Biosciences) and used for plasma selection and PBMC isolation from HC, HS and COPD patients. The cells were isolated using a density gradient centrifugation (Ficoll-paqueTM PLUS; Amersham Biosciences SE-751 84, Uppsala, Sweden) and, after two washes, the cells were suspended in RPMI 1640 cell culture medium (Invitrogen Life Technologies, Italy) supplemented with 10% heatinactivated fetal bovine serum (FBS), 2 mM l-glutamine, 20 mM HEPES, 100 U/ml penicillin, 100 g/ml streptomycin, 5 × 10−5 M 2-ME and 85 g/ml gentamicin. Purity and viability of the cells was tested using trypan blue exclusion. PBMC (1 × 106 cells/ml) recovered from HC, HS and COPD were cultured in the presence of PMA (50 ng/ml final concentration) (Sigma Aldrich) and ionomycin calcium salt (250 ng/ml final concentration) (Sigma Aldrich) for 48 h in 24-well cell culture plates in complete medium RPMI 1640 (Invitrogen Life Technologies, Italy) plus 10% heat-inactivated FBS. Cultured PBMC were assessed for intracellular cytokines, signal transduction and ACh by flow cytometry as described below.
Stimulation of human PBMC from COPD patients with drugs Materials and methods Patients All subjects were patients from the Section of Respiratory Medicine of the Italian National Research Council, Palermo, Italy. The local Ethical Committee approved the study, and participating subjects gave their written informed consent. The study was carried out using samples of three groups of subjects: 13 healthy asymptomatic non-smoking subjects with normal lung function (HC); 12 asymptomatic smokers with normal lung function (HS); 16 patients with chronic obstructive pulmonary disease (COPD). The diagnosis of COPD and the assessment of its severity were defined and classified according to the criteria reported by the Global Initiative for Obstructive Lung Disease (GOLD) guidelines for COPD management (GOLD stage ≥I) (Pauwels et al. 2001). COPD subjects with exacerbations within 1-month prior to the study were excluded. Patients with COPD and HS had a smoking history of 10 pack years or more.
PBMC (1 × 106 cells/ml) from COPD patients (n = 6) were cultured for 48 h in 24-well cell culture plates as previously described, in the presence or absence of the anticholinergic, Tiotropium Spiriva® (100 nM) (Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany) and the 2-AR-agonist, Olodaterol (1 nM) (Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany), alone or in combination. Furthermore, we studied the effect of hemicholinium (HCh-3) (50 M, Sigma–Aldrich), a potent and selective choline uptake blocker, in PBMC from COPD patients cultured for 48 h and compared with the anticholinergic compound Tiotropium (100 nM). The cell viability was evaluated by trypan blue exclusion at the end of each experiments, to exclude the toxicity of the drugs. After PBMC stimulation with drugs, the cells were harvested and stained with fluorescent-labeled antibodies for surface staining, intracellular cytokine, signal transduction and ACh expression and analyzed by flow cytometry as described afterwards.
394
M. Profita et al. / Immunobiology 219 (2014) 392–401
Fig. 1. Gating strategy for flow cytometric identification of lymphocyte subpopulations. The total lymphocytes were gated by forward and side scatter (red colored R1). The lymphocyte gate is further analyzed for its expression of CD3, CD4, CD8 and CD4/CD25 and each positive subpopulations were gated as R2 to allow respectively ulterior targeted analysis.
Stimulation of human PBMC from HC with ACh and CSE PBMC from HC (n = 6) and from COPD (n = 6) were cultured for 48 h in 24-well cell culture plates as previously described were cultured in the presence or absence of ACh (10 M) for 48 h. CSE was prepared as previously described (Profita et al. 2008) and further diluted to the required concentration of 5% in fresh culture medium. PBMC from HC (n = 6) cultured in the same conditions, were treated with and without cigarette smoke extract (CSE) 5% for 24 h, in the presence or absence of Tiotropium (100 nM). The viability of the cells exposed to CSE and Tiotropium was evaluated by trypan blue exclusion dye assay.
After stimulation, the cells were harvested and stained with fluorescent-labeled antibodies for surface staining, intracellular cytokine, signal transduction and ACh expression and analyzed by flow cytometry as described afterwards. Cell surface staining of human PBMC To describe the Peripheral Blood T-cell (PBT) phenotypes in PBMC from HC and HS subjects and from COPD patients, the following fluorochrome conjugated antibodies were used in different combinations: anti-CD3 (HIT3a, BD Pharmingen), anti-CD8 (BW135/80, Miltenyi Biotec Inc, Germany), anti-CD8 (RPA-T8, BD Pharmingen), anti-CD4 (M-T466, Miltenyi Biotec Inc., Germany),
Table 1 Demographic characteristics of patients.
Age (years) Sex, n (Male/Female) FEV1 (% predicted) FVC (% predicted) FEV1/FVC ratio% Smoking pack/yr
HC
HS
COPD
p value
(n = 13)
(n = 12)
(n = 16)
HC vs. HS
HC vs. COPD
HS vs. COPD
60 ± 7 6/7 125.5 ± 26.8 106.6 ± 15.3 88.5 ± 6.4 0
64 ± 3 7/5 105.5 ± 5.0 95.5 ± 3.5 95.0 ± 3.5 45.7 ± 18.3
66 ± 14 8/8 63.7 ± 10.2 70.6 ± 11.2 63.4 ± 7.0 42.0 ± 10.2
ns – ns ns ns O.001
ns –
