Study population
Forty-three patients with sarcoidosis were analyzed. In all cases, the diagnosis was made from a biopsy obtained either from the lungs or from lymph nodes and showing non-caseating epithelioid granulomas with no evidence of inorganic material known to cause granulomatous diseases.
The patients underwent bronchoalveolar lavage (BAL) fluid analysis. In particular, twenty-nine sarcoid patients presenting with an episode of pulmonary involvement were evaluated at the onset of the disease. They were defined as having a high intensity alveolitis (i.e., the active form of the disease) on the basis of the following characteristics: lymphocytic alveolitis (> than 30x103 lymphocytes/ml); lung CD4 to CD8 ratio >4.0. The assessment of disease activity included BAL, clinical features, chest radiograph, lung function tests, high-resolution computed tomography, and routine blood studies. BAL samples were also obtained from fourteen patients with previously diagnosed pulmonary sarcoidosis who repeated BAL fluid analysis during their follow-up period. These patients had normal lung function, normal BAL fluid cell numbers, and no clinical signs of acute disease. No patient received immunosuppressive therapy for 6 months prior to the BAL execution.
Eight subjects were selected as controls for the BAL studies, evaluated for cough complaints without lung disease. They had normal physical examination, chest X rays, lung function tests, and BAL cell numbers.
Peripheral blood from patients with sarcoidosis and from eight healthy subjects was also included in the study. Written informed consent was obtained from each patient and from controls.
Preparation of cell suspensions
Following administration of local anaesthesia, BAL was performed as previously described [20]. Briefly, a total of 150–200 ml of saline solution was injected via fiber-optic bronchoscopy, in 25 ml aliquots, with immediate vacuum aspiration after each aliquot. The fluid was filtered through gauze, and its volume was measured. The amount of injected fluid recovered was 77.3 ± 7.5 %. Cells recovered from the BAL fluid were washed 3 times with PBS, resuspended in endotoxin tested RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 20 mM HEPES and L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 % fetal calf serum (FCS), (ICN Flow, Costa Mesa, CA) and then counted. Alveolar macrophages (AMs), lymphocytes, neutrophils and eosinophils 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 BAL cell suspensions by rosetting with neuraminidase-treated sheep red blood cells followed by Ficoll-Hypaque gradient separations, as previously described [1]. AMs were further enriched by removing residual CD3+, CD16+, and CD56+ lymphocytes with magnetic separation columns (MiniMACS, Miltenyi Biotec), as previously described [1]. Staining with monoclonal antibodies (mAbs) showed that, after this multistep selection procedure, >98 % of AMs expressed the AM-associated CD68 antigen, whereas >98 % of the rosetting population was constituted by CD3+ T cells. CD4+ T cells were separated from CD8+ T lymphocytes by magnetic separations over columns (Mini MACS, Sunnyvale, CA), as previously reported [1].
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. Peripheral blood 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).
Monoclonal antibodies and cytokines
The commercially available conjugated or unconjugated mAbs used belonged to the Becton Dickinson and Pharmingen (San Diego, CA) series and included: CD3, CD4, CD8, CD11c, CD14, CD16, CD19, CD45R0, CD45RA, CD68, IL4 and IFN-γ, and isotype-matched controls. Anti- mAbs were purchased from R&D Systems Inc. (Minneapolis, MN). Purified mouse IgG1 anti-human TL1A (clone 12 F11) was purchased from Human Genome Sciences (GlaxoSmithKline, Verona, Italy) while mouse IgG1 anti-human DR3/TNFRSF25 was from R&D (R&D Systems, Milan, Italy). These mAbs were used for flow cytometry and immunohistochemistry analyses. A secondary antibody PE-conjugated rat anti-mouse IgG1 (Caltag Laboratories, Burlingame, CA, USA) was used for flow cytometry analysis. The frequency of positive cells for TL1A and DR3 was determined by FACS analysis. Cells were acquired on FACSCanto analyzer (Becton-Dickinson) and data processed by FACSDiva software program (Becton-Dickinson).
Confocal microscopy
T cells and AMs from BAL of patients with active (n = 4) and inactive (n = 4) sarcoidosis and controls (n = 3) were plated in polylisine coated glass for 15 min at +4 °C, with anti-TL1A (1:150) and anti-DR3 (1:150) mAbs, and fixed in 4 % paraformaldehyde for 10 min. To reveal positivity for the molecules, a FITC-conjugated rat anti-mouse IgG1 (1:200) was used. Background staining with FITC-conjugated rat anti-mouse IgG1 alone was routinely compared with positively stained cells and was not visible using identical acquisition settings.
Slides were mounted with cover slips and fluorescence was detected using the UltraView LCI confocal system equipped with a fluorescence filter set for excitation at 488 nm.
Western blot analysis
BAL cells (0.25 × 106 for each sample) purified from 8 patients affected by active sarcoidosis, 7 patients with inactive disease, and 8 controls, were prepared by cell lyses with Tris 20 mM, NaCl 150 mM, EDTA 2 mM, EGTA 2 mM, Triton X-100 0.5 % supplemented with complete protease inhibitor cocktail (Roche; Mannheim, Germany) and sodium orthovanadate 1 mM (Calbiochem; Gibbstown, NJ). Samples were then subjected to SDS/PAGE (10 % gels), transferred to nitrocellulose membranes, and immunostained with goat polyclonal Ab anti-human TL1A/TNFS15 (R&D Systems Inc.), mouse mAb anti-human DR3/TNFRSF25 (R&D Systems Inc.), and mouse mAb anti-human β-actin (Sigma-Aldrich), using an enhanced chemiluminescent detection system (Pierce; Rockford, IL).
Immunohistochemical analysis
Lung samples from eight cases of sarcoidosis (five from active, three from inactive forms, the last obtained from native lungs of patients requiring lung transplantation) 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, 1 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 horseradish peroxidase (HRP) polymer (Dako, Glostrup, Denmark) for 30 min. Immunoreactivity was visualized with 3-3’-diaminobenzidine (DAB, Dako, Glostrup, Denmark). Negative controls for non-specific binding were processed omitting the primary antibodies and revealed no signal.
Real-Time PCR expression analysis
Total cellular RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and was treated with DNase (Qiagen). Complementary DNA was generated from 1 μg of total RNA using oligo-dT primer and the AMV reverse transcriptase (Promega, Madison, WI). Real-time PCR was carried out in an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). SYBR Green PCR Master Mix was purchased from Applied Biosystems. Real-Time PCR for TL1A, DR3, MMP-9 and TIMP-1 gene expression was performed in purified AMs and BAL T cells of patients with active (n = 25) and inactive sarcoidosis (n = 13). TL1A and DR3 mRNA levels were also evaluated in monocytes and T cells from the peripheral blood of the same patients and 8 healthy controls. The primers used were: TL1A: forward 5’-CAC CTC TTA GAG CAG ACG GAG ATA A-3’, reverse 5’-TTA AAG TGC TGT GTG GGA GTT TGT-3’; DR3: forward 5’-ACC CAT CTG TCA CCC TTG GA-3’, reverse 5’-CTG GAC GGT GCA GAT CTT CTC-3’; MMP-9: forward 5’-TGC CCG GAC CAA GGA TAC AG-3’, reverse 5'- GTG CAT TCC TCA CAG CCA ACA G; glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward 5’-AAT GGA AAT CCC ATC ACC ATC T-3’; reverse 5’-CGC CCC ACT TGA TTT TGG-3’. The primers were designed in our laboratory, whereas for TL1A we used the primers as described by Migone et al. [21]. Standard curves were generated for each gene. The relative amounts of messenger RNA (mRNA) were normalized for GAPDH expression.
Gelatin zymography assays
This is an in vitro assay using gelatin-substrate gel electrophoresis we employed to measure the level of MMP-9 activity in BAL fluid components or in 24 h culture medium recovered from AMs and lung T cells, of patients with active (n = 7) and inactive (n = 6) sarcoidosis, cultured with or without TL1A (100 ng/ml, R&D Systems). BAL fluid components and conditioned supernatants were mixed with an equal volume of sample buffer (62.5 mM Tris–HCl, pH 6.8, 10 % glycerol, 2 % SDS, and 0.00625 % (w/v) bromophenol blue). Samples were electrophoresed on 7.5 % SDS polyacrylamide gel containing 2 mg/mL gelatin (type A, Sigma-Aldrich). After electrophoresis, the gel was washed three times for 30 min in 2.5 % Triton X-100 at room temperature, and incubated for 16 h at 37 °C in incubation buffer (50 mM Tris–HCl, pH 7.6, 5 mM CaCl2, 200 mM NaCl). The gel was stained for 30 min with Coomassie Brilliant Blue R-250 (Amersham Biosciences) and destained in washing solution (30 % methanol, 10 % acetic acid). White bands on the blue background represented gelatin digestion corresponding to the presence/activity of MMP-9. The bands were quantified by the image analysis software QuantityOne (Bio-Rad).
Statistical analysis
Statistical analysis was performed using Student's t test, Kolgomorov-Smirnov analysis, and ANOVA. Data were expressed as mean ± standard deviation (SD) and were considered statistically significant when p values were <0.05.