Facco et al
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ONLINE ONLY MATERIAL – METHODOLOGY
Preparation of cell suspensions
Following administration of local anesthesia, BAL was performed as previously
described.[16] Alveolar macrophages (AMs), lymphocytes, neutrophils and eosinophils
recovered from the BAL fluid were differentially counted in cytocentrifuged smears stained
with Wright-Giemsa, for a total count of 300 cells, according to morphological criteria.
AMs and BAL T-cells were purified from the BAL cell suspensions by rosetting with
neuraminidase-treated sheep red blood cells followed by Ficoll-Hypaque gradient
separations, as previously described.[17] AMs were further enriched by removing residual
CD3+, CD16+, and CD56+ lymphocytes with magnetic separation columns (MiniMACS,
Miltenyi Biotec), as previously described.[8] Staining with monoclonal antibodies (mAbs)
showed that, after this multistep selection procedure, >98% of AMs expressed the AMassociated CD68 antigen, whereas >98% of the rosetting population was constituted by
CD3+ T-cells.
Peripheral blood mononuclear cells (PBMCs) from the patients under study were obtained
from freshly heparinized blood following centrifugation on Ficoll-Hypaque gradient and
washing with PBS. PB lymphocytes were further enriched following rosetting of PBMC with
sheep-red-blood cells, as reported above, and CD4+ T-cells were separated from CD8+ T
lymphocytes by magnetic separations over columns (Mini MACS, Sunnyvale, CA). After
this selection procedure, more than 97% of the cells were viable and 95% to 99% showed
CD4+ phenotype.
Intracellular cytokine staining
Detection of intracellular cytokines were performed in freshly obtained lung and
peripheral blood cells and in equivalent cells stimulated with 20 ng/ml PMA and 1 mM
ionomycin (Sigma-Aldrich, Milan, Italy), for 4 hours, in the presence of 5 μg/ml Brefeldin
A (Sigma-Aldrich) added for the last 3 hours of culture, stained for surface antigens,
Facco et al
fixed, and permeabilized using Fix&Perm reagent (Caltag Laboratories, Burlingame,
CA) for 20 minutes. After these procedures, anti-IFN- and anti-IL-17 mAbs were added.
The frequency of BAL cells positive for the above reagents was determined by
flow cytometry, as previously described [6]. For direct fluorescence-activated cell sorting
(FACS) analysis, FITC- or PE-conjugated control isotype matched mouse mAbs were
used to set the fluorescence background (IgG1, IgG2a, and IgG2b, Becton Dickinson).
For FACS analysis, 104 cells were acquired and the expression was determined by
overlaying the histograms of the samples stained with the different reagents. Both BAL
lymphocytes and AMs were gated in flow cytometry analysis with two different
approaches: a) physical characteristics of cells, and b) expression of the T-associated
CD3 and CD4, and AM-associated CD11c, CD14, and CD68 antigens on the area of
lymphocytes and AMs, respectively. The purity of the gates was always > 98% of cells.
Cells were scored using a FACSCalibur analyzer (Becton Dickinson), and data were
processed with the CELLQuest software program (Becton Dickinson).
Immunohistochemical analysis
Lung samples from seven cases of sarcoidosis (three from active, two from inactive
forms, and two obtained from native lungs of patients requiring lung transplantation for
the development of lung fibrosis) and lymph node samples from two patients with active
sarcoidosis were processed by immunohistochemistry. Briefly four µm-thick sequential
serial sections were pre-treated by boiling in citrate buffer (pH 6.1) in a microwave (700
W, one minute) for antigen retrieval. Afterwards, sections were treated with normal
serum (Immunotech, Marseille, France) and incubated for 60 min with the primary
monoclonal antibodies anti-IL 17 and anti-IL23R at a concentration of 1:20 and 1:50.
Sections were subsequently incubated with rabbit HRP polymer (Dako, Glostrup,
Denmark) for 30 min. Immunoreactivity was visualized with 3-3’-diaminobenzidine
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(DAB, Dako, Glostrup, Denmark). Negative controls for nonspecific binding were
processed omitting the primary antibodies and revealed no signal.
RNA Purification and Real-Time Polymerase Chain Reaction for the Quantification
of IL-17 and RORγt Expression by Sarcoid Lung Cells
Total RNA was prepared from purified T lymphocytes obtained from patients with
sarcoid T-cell alveolitis or normal T cells or purified alveolar macrophages from patients
with active sarcoidosis and control subjects, as previously described.[6] Total cellular
RNA was extracted from 1.5-5.0x106 sarcoid T lymphocytes or macrophages using
Rneasy Mini Kit (Qiagen, Germany), according to the manufacturer's protocol and
treated with DNase (Qiagen). First strand complementary DNA (cDNA) was generated
from 1 μg total RNA using oligo-dT primer and the AMV reverse transcriptase (Reverse
Transcription System, Promega Corporation, Madison, WI, USA).
Real-time quantitative PCR amplification reactions were carried out in an ABI Prism
7000 sequence detection system (Applied Biosystems, Foster City, CA) in a 15 μl
volume. SYBR Green PCR Master Mix was purchased from Appplied Biosystems (P/N
4309155), containing AmpliTaq Gold DNA Polymerase and optimized buffer
components. A fraction of 5 μM primers and 1,5 μl of cDNA were added to SYBR Green
master mix to make a final 15 μl reaction volume. The primers used for IL-17, RORγt
and GAPDH amplifications were: IL-17: Forward 5’- AAgACCTCATTggTgTCACTgCTA
CTg -3’, Reverse 5’- CTggATTTCgTgggATTgTgATTCCTg -3’; RORγt: Forward 5’- ATg
TCCCgAgATgCTgTCAAgTTC -3’, Reverse 5’- gTCgTCCCTCTgCTTCTTg -3’; GAPDH:
Forward 5’- AATggAAATCCCATCACCATC -3’, Reverse 5’- CgCCCCACTTgATTTTgg 3’. PCR reactions were performed under the following conditions: 10 minutes
denaturation at 95 °C followed by 95 °C 15 seconds, 60 °C 1 minute cycled 50 times.
Each quantitation target was amplified in triplicate sample. A no template control for
each master mix and three standard curves were generated for GAPDH, IL-17 and
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RORγt using total T cell cDNA in a serial dilution 1:1, 1:5, 1:25, and 1:125. The relative
amounts of mRNA were determined by comparison with standard curves. Each sample
results were normalized for GAPDH expression. To distinguish specific amplicons from
non-specific amplifications, a dissociation curve was generated.
Statistical analysis
Normal distribution of the variables of interest was verified with the Kolmogorov-Smirnov
test. Normal data are expressed as mean ± standard deviation (SD). Differences of
normal variables among the three groups (active sarcoidosis, inactive sarcoidosis and
controls) were analyzed by one-way ANOVA followed by post-hoc comparisons with
Bonferroni correction. To compare migration of cells with and without chemokine
stimulation we used a two-tailed unpaired Student’s t test. Data were analyzed with the
assistance of the Statistical Analysis System and statistical significance was accepted
at p<0.05.
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