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Online only Supplement
Increased CCL5 and CXCL7 chemokine expression is associated with neutrophil activation in
severe stable COPD
Antonino Di Stefano, PhD1*, Gaetano Caramori, MD2*, Isabella Gnemmi1, Marco Contoli, MD2, Laura
Bistrot PhD2, Armando Capelli, MD1, Fabio LM Ricciardolo, MD3, Francesca Magno, PhD1, Silvestro
Ennio D’Anna, MD4, Andrea Zanini, MD1, Marco Carbone, PhD1, Federica Sabatini, PhD5, Cesare
Usai, PhD6, Paola Brun, PhD7, Kian Fan Chung8, Peter J Barnes8, Alberto Papi, MD2, Ian Adcock,
PhD8, Bruno Balbi, MD1.
*Both Authors contributed equally to this work.
Author for correspondence:
Antonino Di Stefano, PhD
Fondazione S. Maugeri, IRCCS
Laboratorio di Citoimmunopatologia Apparato Cardio Respiratorio
Via per Revislate 13, 28010 Veruno (NO), Italy
Phone: +39 0322 884711
Fax: +39 0322 884776
Email: antonino.distefano@fsm.it
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Introduction
Pathological studies performed in patients with COPD show that inflammation occurs in the central,
peripheral airways (bronchioles) and lung parenchyma.E1-E4 Some of these studies have emphasized the
potential role of several inflammatory cells in the pathogenesis of COPD including alveolar
macrophages, T-lymphocytes and CD8+ cells. In contrast, fewer studies have investigated neutrophil
granulocytes, despite increased numbers being reported in bronchial mucosa, particularlyof severe
COPD patients,E4-E6 in small airways,E3 bronchoalveolar lavage (BAL) fluidE7,E8 and in sputum
samples.E9 Furthermore, the need for pathologic investigations in patients with more severe COPD has
been underlined.E3 However, the molecular mechanisms responsible for this neutrophilia, particularly
that seen in the tissue,are not fully clarified. Neutrophil prevalence may result from increased
chemotaxis, increased neutrophil adhesion to collagens or other components of the extracellular matrix
or to prolonged survival.E10 Recently, a partial loss of the chemotactic response to CXCL8 and Nformyl-methionyl-leucyl-phenylalanine (fMLP) has been reported for sputum neutrophils from stable
COPD patients, suggesting a hypo-functional status of these cells after migration and residence in the
bronchial lumen.E11 This further emphasises the need to examine the functional status of neutrophils
within the airways of patients with COPD.
Several chemokines of the CXC and CC family are involved in neutrophil chemotaxis. E12-E14
Cysteine-X-cysteine chemokine ligand CXCL1 [growth-related oncogene α (GRO-α,)] and CXCL5
[epithelial-neutrophil activating peptide 78 (ENA-78)] may be involved in neutrophilic inflammation
and can both bind to and activate CXCR2. CXCL1 expression is increased in sputum from patients
with COPD compared to non smokers and healthy smokers.E15 CXCL5 is derived predominantly from
bronchial epithelial cellsE16 and BAL CXCL5 concentrations are enhanced in COPD patients
compared to normal subjects.E17 However, there is no difference in CXCL5 levels between patients
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with emphysema and normal smokers.E17 Finally, a marked increase in CXCL5 gene expression has
been reported in bronchial biopsies during exacerbations of mild/moderate COPD.E18 In humans,
CXCL6 [granulocyte chemotactic protein-2 (GCP-2)] complements the activity of CXCL8 (IL-8) and
induces neutrophil chemotaxis and activationE19 whereas lung macrophages release CXCL6 after
lipopolysaccharide (LPS) stimulation.E20 However, there is a complete absence of data concerning
CXCL6 expression in COPD. CXCL7 [neutrophil-activating peptide-2 (NAP-2)] is also chemotactic
for neutrophils and is released as an inactive precursor by platelets.E21 Increased protease activity
within the lung by enzymes such as cathepsin-G can activate CXCL7 and contribute to its chemotactic
activity.E13,E21 In addition, PBMCs from COPD patients have an increased chemotactic response
towards CXCL7.E22 CXCL8 activates neutrophils via a specific low-affinity G-protein receptor
(CXCR1), coupled to cell activation and degranulation, and via a high-affinity receptor (CXCR2)
shared with other members of the CXC family.E23 CXCL8 levels are elevated in the sputum of patients
with COPD and are correlated with disease severity
E24
and increase further during COPD
exacerbations.E18,E25
The CCR1 ligand CCL5 (RANTES; released by activated normal T cells expressed and secreted) is
significantly increased in the sputum of patients with stable COPD when compared with non-smokers
but not with smokers with normal lung function and its expression correlates with sputum
neutrophilia.E26 Sputum CCL5 concentration is also increased in COPD patients during
exacerbations.E27 In addition, LPS stimulated cultured lung explants from COPD patients showed
increased release of CCL5 compared with explants from control smokers.E28
The leukocyte M2 integrin (also known as Mac-1, complement receptor type 3 (CR3), and
CD11b/CD18) functions as an adhesion molecule facilitating diapedesis. Overexpression of CD11b has
been reported in blood neutrophilsE29,E30 and sputumE31 of stable COPD patients compared to control
subjects. CD62E (E-selectin), the major ligand of CD11b, is increased in bronchial biopsies of patients
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with stable COPD,E32 but the expression of CD11b on neutrophils in the bronchial mucosa of patients
with stable COPD is still unknown.
CD44 belongs to the family of hyaluronan-binding type I transmembrane glycoproteins receptors.
Hyaluronan, an extracellular matrix component, is the main ligand for CD44.E33 Neutrophils cocultured with human primary bronchial epithelial cells and GM-CSF up-regulate CD44.E34 The
expression of CD44 on neutrophils in the bronchial mucosa of patients with stable COPD has not been
previously reported.
Methods
Subjects
All subjects were recruited from the Section of Respiratory Medicine of the Fondazione Salvatore
Maugeri (Veruno, Italy). We examined bronchial biopsies from 49 subjects by immunohistochemistry;
13 subjects were current or ex smokers with normal lung function, 25 had a clinical diagnosis of COPD
and 11 were never-smokers with normal lung function (Table 1). The severity of the airflow
obstruction was staged using GOLD criteria.E1 All former smokers had stopped smoking for at least
one year. COPD and chronic bronchitis were respectively defined, according to international
guidelines, as: the presence of post-bronchodilator forced expiratory volume in one second (FEV1)/
forced vital capacity (FVC) ratio <70% or the presence of cough and sputum production for at least 3
months in each of two consecutive years.E1
All COPD patients were stable with no previous
exacerbation in the 6 months before bronchoscopy. None of the subjects was treated with theophylline,
antibiotics, antioxidants, mucolytics, and/or glucocorticoids in the month prior to the bronchial biopsy.
The study conformed to the Declaration of Helsinki, Ethics Consent was obtained, bronchial biopsies
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were performed according to the local Ethics Committee Guidelines, and informed consent was
obtained from each subject.
Lung function tests and volumes
Pulmonary function tests were performed as previously describedE6 according to published guidelines.
Pulmonary function tests included measurements of FEV1 and FEV1/FVC under baseline conditions in
all the subjects examined (6200 Autobox Pulmonary Function Laboratory; Sensormedics Corp., Yorba
Linda, CA). Predicted values for the different measures were those from the Communité Européenne
du Carbon et de l’Acier.
In order to assess the reversibility of airflow obstruction and
postbronchodilator functional values, the FEV1 and FEV1/FVC% measurements in the groups of
subjects with FEV1/FVC%70% pre-bronchodilator was repeated 20 min after the inhalation of 0.4 mg
of salbutamol.
Fiberoptic bronchoscopy, collection and processing of bronchial biopsies
Subjects attended the bronchoscopy suite at 8.30 am after having fasted from midnight and were pretreated with atropine (0.6 mg IV) and midazolam (5-10 mg IV). Oxygen (3 l/min) was administered via
nasal prongs throughout the procedure and oxygen saturation was monitored with a digital oximeter.
Using local anaesthesia with lidocaine (4%) to the upper airways and larynx, a fiberoptic bronchoscope
(Olympus BF10 Key-Med, Southend, UK) was passed through the nasal passages into the trachea.
Further lidocaine (2%) was sprayed into the lower airways, and four bronchial biopsy specimens were
taken from segmental and subsegmental airways of the right lower and upper lobes using size 19
cupped forceps. Bronchial biopsies for immunohistochemistry and RT-QPCR were gently extracted
from the forceps and processed for light microscopy as previously described.E6 Two samples were
embedded in Tissue Tek II OCT (Miles Scientific, Naperville, IL), frozen within 15 min in isopentane
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pre-cooled in liquid nitrogen, and stored at –80°C. The best frozen sample was then oriented and 6m
thick cryostat sections were cut for immunohistochemical light microscopy analysis and 30m thick
cryostat sections were cut for RT-QPCR and processed as described below. Bronchial biopsies for
western blot analysis were immediately placed on ice, frozen in liquid nitrogen and processed as
described below.
Immunohistochemistry
Two sections were stained with immunohistochemical methods. The best immunostained section was
then selected for quantitative purposes. The following panel of antibodies was used (Table 2). Briefly,
after blocking non-specific binding sites using serum derived from the same animal species as the
secondary antibody, primary antibodies were applied at optimal dilutions in TRIS-buffered saline (0.15
M saline containing 0.05 M TRIS-hydrochloric acid at pH 7.6) and incubated (1 hr) at room
temperature in a humidified chamber. Antibody binding was demonstrated with the use of secondary
antibodies anti mouse (Vector, BA 2000), anti rabbit (Vector, BA 1000) or anti goat (Vector, BA 5000)
followed by Strept AB Complex/AP (Dako, K0391) and fast-red substrate. Control slides were
included in each staining run using human tonsil or nasal polyp as a positive control for all
immunostaining performed. For the negative control slides, normal goat or rabbit non-specific
immunoglobulins (Santa Cruz Biotechnology) were used at the same protein concentration as the
primary antibody.
Immunofluorescence staining with confocal microscopy
TableE1 shows the clinical details of the subjects used for the confocal microscopy (n=8).
Sections were fixed with 4% paraformaldehyde, washed with phosphate buffered saline (PBS) and
incubated (1 hour) with PBS containing 5% bovine serum albumin and 5% goat serum. After blocking,
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sections were incubated 1 hour with the primary antibodies diluted 1:50 in PBS containing 5% bovine
serum albumin. The following antibodies were used: rabbit anti-human CD11b (sc-28664; Santa Cruz);
rabbit anti-human CD44 (sc-7946; Santa Cruz) and mouse antihuman neutrophil elastase (M752;
Dako). After washing with PBS, the preparations were incubated for a further 30 min with the
appropriate secondary Alexa Fluor 488- or Alexa Fluor 647 conjugated antibodies diluted 1:200 in
PBS. Negative controls included irrelevant mouse and rabbit immunoglobulins revealed as for primary
antibodies. Slides were mounted using a specific mounting medium (Fluka 10979, Sigma, Italy).
Scoring system for immunohistochemistry and confocal microscopy
Morphometric measurements were performed with a light microscope (Leitz Biomed, Leica
Cambridge, UK) connected to a video recorder linked to a computerised image system (Quantimet 500
Image Processing and Analysis System, Software Qwin V0200B, Leica). Light-microscopic analysis
was performed at a magnification of 630x. Immunostained cells were quantified in the area 100 m
beneath the epithelial basement membrane in several non-overlapping high power fields until all the
available area was covered. The final result, expressed as the number of positive cells per square
millimeter, was calculated as the average of all the cellular counts performed in each biopsy. We
quantified the immunostained cells with at least a portion of the nucleus seen close to
immunopositivity [E35]
The immunostaining for all the antigens studied was also scored (range: 0: absence of immunostaining,
1: from 1% until 33% of immunostained cells, 2: from 34% until 66% of immunostained cells, 3:
extensive (from 67% to 100%) intense immunostaining) in the intact (constituted by columnar and
basal epithelial cells) bronchial epithelium, as previously described.E32 The final result was expressed
as the average of all scored fields performed in each biopsy. A mean  SD of 0.7000.260 millimeters
of epithelium was analyzed in COPD patients and control subjects.
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The slides for confocal microscopy were analyzed using a three-channel Leica TCS SP2 laser scanning
confocal microscope. The Leica LCS software package was used for acquisition, storage, and
visualization. The quantitative estimation of co-localized proteins was performed calculating the “colocalization coefficients”.E36, E37
Quantification of chemokines and cytokines mRNA levels in bronchial biopsies
Table E2 shows the clinical details of the subjects used for the real time RT-PCR (n=31).
Total RNA was extracted (Micro RNeasy Kit, Qiagen Milan, Italy) from 30m thick cryostat sections
of bronchial biopsies and 1 µg used for cDNA synthesis. cDNA was synthesised using Omniscript RT
kit (Qiagen) as per manufacturer’s instructions. Primer pairs for CCL5 (Cat. # QT00090083), CXCL7
(Cat. # QT00001372), CXCL8 (Cat. # QT00000322) were purchased from Qiagen. Quantitative realtime reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out using Sybr-green
(QuantiFast Sybr Green PCR kit cat. # 204054 – Qiagen) following the manufacturer’s protocol. At the
end of the RT-PCR run a melting curve analysis was carried out to verify that the cycle threshold (Ct)
values were based upon a single PCR product (data not shown). Relative levels of cDNAs were
established using the Ct methods against the housekeeping gene guanine nucleotide binding protein
(G protein) (GNB2L Cat. # QT01156610 – Qiagen). After normalization the value of Ct was
subtracted from 45 (total number of RT-PCR cycles), thus higher Ct levels indicate higher mRNA
levels. Also relative levels of mRNAs were expressed as the ratio of the Ct value for the gene of
interest Ct/housekeeping gene Ct [guanine nucleotide binding protein (G protein) - GNB2L Cat. #
QT01156610 from Qiagen].
Western blot analysis for CXCL7, CXCL8 and CCL5 in the bronchial biopsies
Table E3 shows the clinical details of the subjects used for the western blotting (n=12).
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Whole cell proteins were extracted from bronchial biopsies as previously described. E6 In brief, frozen
bronchial biopsies were resuspended with mechanical disruption in RIPA lysis buffer with a protease
inhibitor cocktail immediately frozen to –70 C and thawed after at least 60 minutes. Particulate matter
was removed by centrifugation at 12000 x g for 10 min at 4C. Protein concentration was measured in
the supernatant by the Bradford method according to the manufacturer’s instructions (Bio-Rad
Laboratories, Hemel Hempstead, UK). An equal volume of Laemmli sample buffer 2X concentrate was
added to the final volume of the sample. At least 50 g/lane of whole-cell proteins were subjected to a
4-20% SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose filters (Hybond-ECL,
Amersham Pharmacia Biotech) by blotting. Filters were blocked for 45 minutes at room temperature in
Tris-buffered saline (TBS), 0.05% Tween 20, 5% non-fat dry milk. The filters were then incubated
with goat anti-human CCL5 (AF-278-NA; from R& D Systems) or goat anti-human CXCL7 (sc19224; from www.scbt.com) or goat-anti human CXCL8 (AF-208-NA; from R & D Systems) for 1h at
room temperature in TBS, 0.05% Tween 20, 5% non-fat dry milk at dilution of 1:500at a concentration
of 0.1 mg/ml (CCL5 and CXCL8) or 0.2 mg/ml (CXCL7). As positive controls 50 ng of human
recombinant
CCL5
(278-RN;
from
R
&
D
Systems),
human
recombinant
CXCL7
(www.peprotech.com; cat 300-14) or human recombinant CXCL8 (hBA-72) (sc-4600; from
www.scbt.com) were used. Filters were washed three times in TBS, 0.5% Tween 20 and then incubated
for 45 minutes at room temperature with rabbit anti-goat antibody conjugated to horseradish peroxidase
(Dako, Ely, UK) in TBS, 0.05% Tween 20, 5% non-fat dry milk, at a dilution of 1:4000. After three
further washes in TBS, 0.05% Tween 20 visualization of the immunocomplexes was performed using
the ECL as recommended by the manufacturer (Amersham Pharmacia Biotech). As an internal control
we reprobed each filter with an anti-human actin antibody (Santa Cruz Biotechnology). The 43kDa
(actin) or 8kDa (CCL5 and CXCL8) and 14kDa (CXCL7) bands were quantified using densitometry
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with Grab-It and VisionWorks LS software (UVP, Cambridge, UK) and expressed as a ratio with the
corresponding actin optical density value of the same lane.
Data analysis
Group data were expressed as mean  standard error for functional data or median (range) for
morphologic data. We tested for a normal distribution for functional data (i.e. FEV1%, FVC, age etc.)
and for a non normal distribution for morphologic parameters. Then we applied the analysis of variance
(ANOVA) in comparing subgroups of patients and control subjects for functional data. The non
parametric Kruskal Wallis test was applied for multiple comparisons, without application of Bonferroni
correction, when morphologic data were analysed followed by the Mann-Whitney U test for
comparison between groups. The statistical Guide to GraphPad Prism recommends that the Bonferroni
correction should not be used when comparing more than 5 variables due to the conservative nature of
the test and the subsequent likeliness of missing real differences. We believe that this comparative
analysis is of value and represents part of our informative findings. For this reason we applied specific
non parametric statistical tests to our data of Table 3 and 4 without including the Bonferroni correction.
To verify the degree of association between functional or morphological parameters, in all smokers
with and without COPD or in smokers with COPD alone the correlation coefficients between
functional-morphological and morphological-morphological data were calculated using the Spearman
rank method. Probability values of p<0.05 were considered significant. Data analysis was performed by
using the Stat View SE Graphics program (Abacus Concepts Inc., Berkeley, CA-USA).
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E-Tables
Table E1. Characteristics of subjects for the confocal immunohistochemical study
Subjects
n Age
Sex Smoking History Pack-years FEV1 Pre
M/F
(ex/current)
% pred
Smokers with normal
lung function
Moderate/severe
COPD
4 563 3/1
0/4
383
955
4 654 4/0
3/1
356
345
FEV1 FEV1/FVC
Post
%
% pred
ND
834
406
455
Data are presented as meanSE. COPD: chronic obstructive pulmonary disease; M: male; F: female;
FEV1: forced expiratory volume in one second; FVC: forced vital capacity. ANOVA, p<0.0001
significantly different from smokers with normal lung function; ND=not determined. For COPD
patients FEV1/FVC% are post-bronchodilator values.
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Table E2. Characteristics of subjects used for the RT-QPCR study
Subjects
n
Age
Sex
M/F
Smokers with normal lung
function
Mild/Moderate COPD
7
58.7±4.2
73.3±1.7^
15 67.2±2.0
Severe/very-severe COPD
9
5/2
Smoking History
Pack-years
(Ex/current)
45.7±12 (0/7)
FEV1 Pre
% pred
FEV1 Post
% pred
FEV1/FVC
%
95.43 ±5.1
ND
7/2
44.2±6.7 (4/5)
65.78±3.3
67.33±4.0
56.06±3.0
15/0
60.9±11.1 (12/3)
37.±1.6
40.73±2.0
46.5±2.3§
80.0±2.1
Data are presented as mean  SE. COPD: chronic obstructive pulmonary disease; M: male; F: female;
FEV1: forced expiratory volume in one second; FVC: forced vital capacity. ANOVA, p<0.0001
significantly different from smokers with normal lung function or from mild/moderate COPD;
§
p=0.0049 significantly different from mild/moderate COPD; ^p=0.048 significantly different from
smokers with normal lung function. ND= not determined. For COPD patients FEV1/FVC% are postbronchodilator values.
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Table E3. Characteristics of the subjects used for the western blotting study
Subjects
Smokers with normal lung
function
Moderate/severe COPD
n
5
7
Age
Sex
64.5±4.3 5/0
592
7/0
SmokingHistory
pack-years
(ex/current)
62±14 (2/3)
FEV1 pre
(%)
FEV1 post
(%)
96.2 ±3.7
ND
7015 (2/5)
414
435
FEV1/FVC
%
82±3.4
433
Data are presented as mean  SE. COPD: chronic obstructive pulmonary disease; M: male; F: female;
FEV1: forced expiratory volume in one second; FVC: forced vital capacity. ANOVA, p<0.0001
significantly different from smokers with normal lung function; ND=not determined. For COPD
patients FEV1/FVC% are post-bronchodilator values.
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