Bronchoscopy was performed with a flexible bronchoscope (Pentax, Tokyo, Japan) according to the Polish Respiratory Society Guidelines (Chciałowski et al. 2011). Patients optionally received midanium and atropine before the examination, 2 % lidocaine was used as a topical anaesthetic. BAL fluid (BALF) was collected from medial lobe, by instillation and subsequent withdrawal of 4 × 50 mL of 0.9 % NaCl. The fluid recovery was 52.1 ± 1.2 %. The crude BALF was filtered through a gauze, to clear the thick mucus and other contaminants, next centrifuged, and the pellet was suspended in a phosphate buffer. The total number of non-epithelial cells (total cell count – TCC) was presented as n × 10⁶. Cytospin slides were prepared and stained by May-Grünwald-Giemsa stain. The number of macrophages, lymphocytes, neutrophils, and eosinophils was calculated under a light microscope and presented as % of TCC. After the calculations, all fluid was centrifuged (10 min 1,200 rpm), supernatant of BALF was suspended in RNAlater RNA Stabilization Reagent (Qiagen, Hilden, Germany) in a volume of about 350 μl of solution in Eppendorf tubes, marked with an identification number, and was frozen (−80 °C) until further RNA isolation procedures.

Spirometry was performed according to the Polish Respiratory Society Guidelines (2006) with a computer-based spirometer (Jaeger, Dortmund, Germany). Forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV₁) were measured, and the Tiffenau index (FEV₁/FVC) was calculated. Lung diffusion capacity for carbon monoxide was measured in patients with lung parenchymal disease only (stage II–IV) with a single breath method, with a Lungtest 1000 SB (MES, Cracow, Poland) according to ATS/ERS standards (European Respiratory Society 1993). The values were corrected for the hemoglobin concentration (DLCOc). All data (except the Tiffeneau index) were presented as % of predicted value.

Blood was collected into 2 mL EDTA containing tubes (labeled with the identification number). For lymphocyte separation, a density gradient cell separation medium Histopaque-1077 (Sigma-Aldrich, Poznan, Poland) was used. Blood (3 mL) was carefully layered onto 3 mL of Histopaque-1077 in a 15-mL conical centrifuge tube. Next, samples were centrifuged at 400 × g for 30 min at room temperature. After centrifugation, mononuclear cells were transferred into a clean conical centrifuge tube. Cells were washed by adding 10 mL of isotonic phosphate buffer and mixed gently, then centrifuged at 250 × g for 10 min. The supernatant was discarded. Cells were resuspended in 5 mL of isotonic phosphate buffered saline solution and centrifuged at 250 × g for 10 min. The supernatant was discarded and cells were resuspended in 350 μl RNAlater RNA Stabilization Reagent and frozen (−80 °C).

RNA isolation was performed using mirVana™ miRNA Isolation Kit (Life Technologies, Carlsbad, CA), according to the manufacturer’s protocol. The quality and quantity of isolated RNA was spectrophotometrically assessed (Eppendorf BioPhotometrTM Plus, Eppendorf, Hamburg, Germany). The purity of total RNA (ratio of 16S to 18S fraction) was determined in the automated electrophoresis using RNA 6000 Pico LabChipplates on Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

cDNA was transcribed from 100 ng of total RNA, using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA) in a total volume of 20 μl. Reverse transcription (RT) master mix contained the following: 10 x RT buffer, 259 dNTP Mix (100 mM), 10 x RT Random Primers, MultiScribeTM Reverse Transcriptase, RNase Inhibitor, and nuclease-free water. RT reaction was performed in a personal thermocycler (Eppendorf, Hamburg, Germany) in the following conditions: 10 min at 25 °C, followed by 120 min at 37 °C; then the samples were heated to 85 °C for 5 s, and held at 4 °C. The relative expression analysis was performed in 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA) using TaqMan probes for the studied genes: TGF-β₁ (Hs00998133_m1), SMAD2 (Hs00183425_m1), SMAD3 (Hs00969210_m1), SMAD7 (Hs00998193_m1). The PCR mixture contained: cDNA (1–100 ng), 20 × TaqManR Gene Expression Assay, 2 × KAPA PROBE Master Mix (2x) ABI Prism Kit (Kapa Biosystems, Wilmington, MA) and RNase-free water in a total volume of 20 μl. The expression levels (RQ values) of the studied genes were calculated using the delta delta CT method, with the adjustment to the β-actin expression level and in relation to the expression level of calibrator (Human Lung Total RNA Ambion®), for which RQ value was equal to 1.

The Kruskal–Wallis test, the Mann–Whitney U test, the Neuman–Keuls’ multiple comparison test, and the Spearman rank correlation were used to assess the correlation between the relative gene expression levels and sarcoidosis groups classified on the basis of chest X-ray results (stage I vs. II–IV) and disease phenotype (acute vs. insidious onset), spirometric parameters, DLCOc, serum Ca²⁺ concentration, Ca²⁺ in 24 h urine collection, the percentage of lymphocytes in BAL, the phenotype of immune cells (CD4⁺/CD8⁺), age, and sex of patients. P < 0.05 was considered statistically significant.