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 N-

formyl-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 with

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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 M2 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 co-

cultured 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 pre-

treated 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 6m

thick cryostat sections were cut for immunohistochemical light microscopy analysis and 30m 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 0.7000.260 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 “co-

localization 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 30m 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 real-

time 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 4C. 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 (sc-

19224; 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 SexM/F

Smoking History(ex/current)

Pack-years FEV1 Pre% pred

FEV1

Post% pred

FEV1/FVC%

Smokers with normal lung function

4 563 3/1 0/4 383 955 ND 834

Moderate/severe COPD

4 654 4/0 3/1 356 345 406 455

Data are presented as meanSE. 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/FSmoking History

Pack-years(Ex/current)

FEV1 Pre% pred

FEV1 Post% pred

FEV1/FVC%

Smokers with normal lung function

7 58.7±4.2 5/2 45.7±12 (0/7) 95.43 ±5.1 ND 80.0±2.1

Mild/Moderate COPD 9 73.3±1.7^ 7/2 44.2±6.7 (4/5) 65.78±3.3 67.33±4.0 56.06±3.0

Severe/very-severe COPD 15 67.2±2.0 15/0 60.9±11.1 (12/3) 37.±1.6 40.73±2.0 46.5±2.3§

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 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 post-bronchodilator values.

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Table E3. Characteristics of the subjects used for the western blotting study

Subjects n Age Sex SmokingHistorypack-years(ex/current)

FEV1 pre(%)

FEV1 post(%)

FEV1/FVC%

Smokers with normal lung function

5 64.5±4.3 5/0 62±14 (2/3) 96.2 ±3.7 ND 82±3.4

Moderate/severe COPD 7 592 7/0 7015 (2/5) 414 435 433

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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