Supplemental Methods - 1 -
Supplemental Methods
Inclusion and Exclusion Criteria
Healthy nonsmokers
Inclusion criteria
$ Must be capable of providing informed consent
$ Males and females, age 18 or older
$ Never-smokers by history, with current smoking status validated by the absence of nicotine
metabolites in urine
$ Good overall health without history of chronic lung disease, including asthma, and without
recurrent or recent (within 3 months) acute pulmonary disease, including chronic or acute
bronchitis
$ Normal physical examination
$ Normal routine laboratory evaluation, including general hematologic studies, general
serologic/immunologic studies, general biochemical analyses, and urine analysis
$ Negative HIV serology
$ Normal chest X-ray (PA and lateral)
$ Normal electrocardiogram (sinus bradycardia, premature atrial contractions are permissible)
$ Females - not pregnant
$ No history of allergies to medications to be used in the bronchoscopy procedure
$ Not taking any medications relevant to lung disease or having an effect on the airway
epithelium
$ Willingness to participate in the study
Exclusion criteria
Unable to meet the inclusion criteria
Pregnancy
Current active infection or acute illness of any kind
Current alcohol or drug abuse
Evidence of malignancy within the past 5 years
Healthy smokers
Inclusion criteria
$ Must be capable of providing informed consent
$ Males and females, age 18 or older
$ Current daily smokers with any number of pack-yr, validated by any of the following: urine
nicotine >30 ng/ml or urine cotinine >50 ng/ml
$ Good overall health without history of chronic lung disease, including asthma, and without
recurrent or recent (within 3 months) acute pulmonary disease
$ Normal physical examination
$ Normal routine laboratory evaluation, including general hematologic studies, general
serologic/immunologic studies, general biochemical analyses, and urine analysis
$ Negative HIV serology
Supplemental Methods - 2 -
$ Normal chest X-ray (PA and lateral)
$ Normal electrocardiogram (sinus bradycardia, premature atrial contractions are permissible)
$ Females - not pregnant
$ No history of allergies to medications to be used in the bronchoscopy procedure
$ Not taking any medications relevant to lung disease or having an effect on the airway
epithelium
$ Willingness to participate in the study
Exclusion criteria
$Unable to meet the inclusion criteria
$Pregnancy
$Current active infection or acute illness of any kind
$Current alcohol or drug abuse
$Evidence of malignancy within the past 5 years
Smokers with COPD
Inclusion criteria
$ Must be capable of providing informed consent
$ Males and females, age 18 or older
$ Current daily smokers with any number of pack-yr, validated by any of the following: urine
nicotine >30 ng/ml or urine cotinine >50 ng/ml
$ Meeting GOLD stages I-III criteria for chronic obstructive lung disease (COPD) based on postbronchodilator spirometry
$ Taking any or no pulmonary-related medication, including beta-agonists, anticholinergics, or
inhaled corticosteroids
$ Normal routine laboratory evaluation, including general hematologic studies, general
serologic/immunologic studies, general biochemical analyses, and urine analysis
Females - not pregnant
Negative HIV serology
$ Normal electrocardiogram (sinus bradycardia, premature atrial contractions are permissible)
$ All individuals have chest X-ray (PA and lateral) and chest CT
$ No history of allergies to medications to be used in the bronchoscopy procedure
$ Willingness to participate in the study
Exclusion criteria
Unable to meet the inclusion criteria
Individuals in whom participation in the study would compromise the normal care and
expected progression of their disease
Current active infection or acute illness of any kind
Current alcohol or drug abuse
Evidence of malignancy within the past 5 years
Supplemental Methods - 3 -
Sampling Small Airway Epithelium
Flexible bronchoscopy was used to collect 10th to 12th generation small airway epithelial
cells by brushing the epithelium as previously described. Cells were detached from the brush by
flicking into 5 ml of ice-cold LHC8 medium (GIBCO, Grand Island, NY). An aliquot of 0.5 ml
was used for differential cell count (typically 2x104 cells per slide). The remainder (4.5 ml) was
processed immediately for RNA extraction. The number of cells recovered by brushing was
determined by counting on a hemocytometer. To quantify the percentage of epithelial and
inflammatory cells and the proportions of basal, ciliated, secretory and undifferentiated cells
recovered, cells were prepared by centrifugation (Cytospin 11, Shandon Instruments, Pittsburgh,
PA) and stained with Diff-Quik (Baxter Healthcare, Miami, FL), and differential cell counts
were performed. Samples were confirmed to be bona fide small airway samples by expression of
genes encoding surfactant proteins and Clara cell secretory protein as previously described [1].
cDNA Preparation and Microarray Processing
Total RNA was extracted using a modified version of the TRIzol method (Invitrogen,
Carlsbad, CA), in which RNA is purified directly from the aqueous phase (RNeasy MinElute
RNA purification kit, Qiagen, Valencia, CA), yielding 2 to 4 g RNA per 106 cells. RNA samples
were stored in RNA Secure (Ambion, Austin, TX) at -80C. RNA integrity was determined by
assessing an aliquot of each RNA sample on an Agilent Bioanalyzer (Agilent Technologies, Palo
Alto, CA). A NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Wilmington, DE)
was used to determine the concentration of RNA. Double stranded cDNA was synthesized from
1 to 2 g of total RNA using the GeneChip One-Cycle cDNA Synthesis Kit, followed by cleanup
with GeneChip Sample Cleanup Module, in vitro transcription reaction using the GeneChip IVT
Labeling Kit, and cleanup and quantification of the biotin-labeled cRNA yield by
spectrophotometric analysis (all kits from Affymetrix, Santa Clara, CA). All HG-U133 Plus 2.0
Supplemental Methods - 4 -
microarrays were processed according to Affymetrix protocols, hardware and software, including
being processed by the Affymetrix fluidics station 450 and hybridization oven 640 and scanned
with an Affymetrix Gene Array Scanner 3000 7G. Overall microarray quality was verified by the
following criteria: (1) 3'/5' ratio for GAPDH 3 and (2) scaling factor 10.0 [2]. The captured
image data from the HG-U133 Plus 2.0 arrays was processed using MAS5 algorithm (Affymetrix
Microarray Suite Version 5 software). MAS5-processed data was normalized using GeneSpring
version 7.3.1 (Agilent technologies) by setting measurements <0.01 to 0.01, per array, by
dividing the raw data by the 50th percentile of all measurements, and, for identification of
differentially expressed genes, additionally per gene, by dividing the raw data by the median
expression level for all the genes across all arrays in a data set.
TaqMan Real-time PCR
cDNA was synthesized from 2 g RNA isolated from the SAE in a 100 l reaction
volume, using the TaqMan Reverse Transciptase Reaction Kit (Applied Biosystems, Foster City,
CA) with random hexamers as primers. Two dilutions of 1:10 and 1:100 were made from each
sample and duplicate wells were run with each dilution. TaqMan PCR reactions were carried out
using pre-made kits from Applied Biosystems and 2 l of cDNA were used in each 25 l reaction
volume. The PCR reactions were run in an Applied Biosystems Sequence Detection System
7500 and relative expression levels were determined using the Ct method using ribosomal
protein S18 (RPS18) gene as endogenous control. TaqMan gene expression assays (all from
Applied Biosystems) were assessed for selected TJ genes [CLDN1 (Hs01076359_m1), CLDN8
(Hs00273282_s1), CLDN10 (Hs01075312_m1), CGN (Hs00430426_m1)], selected AJ genes
[CDH1 (Hs00170423_m1), CDH2 (Hs00983062_m1), PVRL3 (Hs00210045_m1)], putative AJC
regulating gene FOXA2 (Hs00232746_m1), PTEN pathway genes PTEN (Hs02621230_s) and
FOXO3A (Hs00818121_m1), and 2 known smoking-responsive oxidative stress-related genes
Supplemental Methods - 5 -
cytochrome P450, family 1, subfamily A, polypeptide 1 and cytochrome P450, family 1,
subfamily B, polypeptide 1 (CYP1A1; Hs00153120_m1; CYP1B1; Hs00164383_m1) and
NAD(P)H dehydrogenase, quinone 1 (NQO1; Hs00168547_m1).
Supplemental Methods - 6 -
Supplemental Figure Legends
Supplemental Figure 1. TaqMan real-time PCR validation of the differential expression of
selected AJC-related genes (TJ genes CLDN1, CLDN8, and CLDN10; AJ genes PVRL3, CDH1,
CDH2; putative AJC-regulating gene FOXA2) in the SAE of healthy nonsmokers (n=6) and
healthy smokers (n=6); Data are shown as fold-changes (healthy smokers vs healthy
nonsmokers) of gene expression assessed by TaqMan PCR (black bars) in parallel with
microarray data (white bars). See Table I for gene names. *p<0.05; **p<0.01; ***p<0.005.
Supplemental Figure 2. Immunofluorescence analysis of differential expression of A. CLDN1
and B. CDH1 proteins (red color) in the cytospin preparations of SAE of healthy smokers (n=3)
vs healthy nonsmokers (n=3). Nuclei stained with DAPI (blue color); scale bar -10 m.
Supplemental Figure 3. Principal component analysis (PCA)-based separation of healthy
nonsmokers (green circles; n=53), healthy smokers (orange circles; n=59) and COPD smokers
(blue circles; n=23) using the following gene datasets: A. all genes with P call at least 20%
(n=32,436 probe sets); B. 54 AJC-encoding genes; C. 54 randomly selected oxidative stressrelated genes [4]; and (D-F) three datasets composed of 54 genes randomly selected from all
genes with P call at least 20% (randomly selected genes are listed in Supplemental Table 4).
Supplemental Figure 4. Progressive down-regulation of AJC gene expression in the SAE with
the development of COPD. Log2-transformed normalized expression levels for selected
physiological (CLDN1, CLDN8, CXADR, TJP1, CGN, CDH1) and cancer-associated AJCencoding genes (CDH2, CLDN10, CLDN7) are plotted for all healthy nonsmokers (n=53; green
triangles), healthy smokers (n=59; orange triangles), and COPD smokers (n=23; blue triangles).
p values are indicated. N.S., non-significant.
Supplemental Methods page - 7 -
Reference List
1. Harvey BG, Heguy A, Leopold PL, Carolan BJ, Ferris B, Crystal RG (2007) Modification
of gene expression of the small airway epithelium in response to cigarette smoking. J Mol
Med 85: 39-53.
2. Raman T, O'Connor TP, Hackett NR, Wang W, Harvey BG, Attiyeh MA, Dang DT, Teater
M, Crystal RG (2009) Quality control in microarray assessment of gene expression in
human airway epithelium. BMC Genomics 10: 493.
3. Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P, Fukuchi Y, Jenkins C,
Rodriguez-Roisin R, van WC, Zielinski J (2007) Global strategy for the diagnosis,
management, and prevention of chronic obstructive pulmonary disease: GOLD executive
summary. Am J Respir Crit Care Med 176: 532-555.
4. Carolan BJ, Harvey BG, Hackett NR, O'Connor TP, Cassano PA, Crystal RG (2009)
Disparate oxidant gene expression of airway epithelium compared to alveolar macrophages
in smokers. Respir Res 10: 111.