Subjects, bronchoalveolar lavage and sampling of lung tissue
Patients (n = 9) suffering from mild asthma and definite bronchial hyperresponsiveness were included in the study, reviewed and approved by the Swedish Research Ethical Committee (LU339-00). These patients were 25–31 years of age with positive phadiotope staining, non-smokers, PD20 < 2 mg/ml of methacholine stimulation, displayed stable mild persistent asthmatic conditions with normal spirometry baseline, free of infections six weeks before bronchoscopy, and had no corticosteroid treatment for six months prior to the study. All of the patients with asthma were atopic and sensitive to pets (cat allergy). Five of the patients with asthma had perennial allergy and one patient had seasonal allergy only (birch pollen). The patients with asthma were further divided into two groups based on whether they displayed the previously described BALF fibroblasts or not (n = 5 and 4, respectively).
The control subjects used (n = 5) were 24–51 years of age. These subjects were non-asthmatic healthy volunteers with a reversibility in FEV1 < 12%, with no allergic symptoms, and who did not respond to doses of methacholine lower than 2 mg/ml. BAL was performed by instillation of buffered saline solution with a recovery of more than 70%. Bronchial biopsies were taken from the central airways of the right lung and collected as previously described [17]. Five biopsies were taken from each subject. The biopsies collected did not show any differences in size, vascularity, or muscle content. From each individual 16 sections were analyzed.
Cell cultures and cytospin analysis
Primary fibroblasts-like cells were established from BALF as previously described [18]. Briefly, BALF was centrifuged at 500 g for 10 min and the supernatant was discarded and the precipitated BALF cells were washed with DMEM. The precipitated cells were cultured in DMEM supplemented with 10% Fetal Clone III (Hyclone, UT), 1% L-glutamine, 0.5% gentamicin and 5 μg/ml amfoterracin, until outgrowth of fibroblast-like cells reached confluence (20–30 days). These fibroblast-like cells were characterized by expression of α-SMA, collagen, and high production of proteoglycans (16). The cultures were trypsinized and used in passage 4–7. Cytospin was performed using a cytocentrifuge (Shandon Southern Products Ltd, Runcorn, Cheshire, UK) where cells in the BALF were collected. The cells were stained using the May-Grünwald/Giemsa (Sigma Aldrich, Stockholm, Sweden) method according to instructions from the manufacturer. 200 cells were counted and granulocytes were identified according to their morphological structure.
Histological studies and measurement of basement membrane thickness
Bronchial biopsies were fixed in 4% paraformaldehyd, embedded in paraffin and sectioned (5 μm). Basic morphological characterization was made using Gomoris Trichrome staining kit (Polysciences Inc, Warrington, PA) for staining of nuclei, muscles fibers and collagen I. Photographs were taken using a light microscope camera (DM IRB, Leica, Wetzlar, Germany).
Basement membrane thickness was quantified by measuring the area of the entire basement membrane, from the base of the bronchial epithelium to the outer limit of the lamina reticularis, and the length of the true basement membrane. This was performed using a computerized image (Leica Software Q500IW, Leica, Wetzlar, Germany).
Thickness was calculated as area/length of the basement membrane. These measurements were performed as previously described [19].
Confocal microscopy
Embedded and fixed tissue sections (5 μm) and cultured BALF fibroblasts were dehydrated and incubated with primary monoclonal mouse anti-human antibodies: CD34, CD45RO (BD Biosciences, Pharmingen, Leiden, The Netherlands) and α-SMA with Cy3 conjugates (Sigma Aldrich, Stockholm, Sweden). For cultured BALF fibroblast, cells were fixed in 4% paraformaldehyde and 0.5% Triton X-100 in PBS was used as a permeabilization agent prior α-SMA staining. Secondary antibodies used for detection of CD34 and CD45RO were Alexa Fluor 488 and 647 (Molecular Probes, Eugene, OR), respectively. CD45RO and prolyl-4-hydroxylase detection; fixed tissue sections were dehydrated, treated with 1% trypsin, covered with blocking solution (PBS containing 3%BSA, 5% fatfree milkpowder, and 1% goat serum) and incubated with primary monoclonal antibody CD45RO (BD Biosciences, Pharmingen, Leiden, The Netherlands), prolyl-4-hydroxylase (Dakocytomation, Denmark) and nucleic staining (Sytox Blue Nucleic Acid Stain, Molecular Probes, Eugene, OR). Sections were then washed with TBS+0.5% Triton X. Secondary antibodies used for detection of CD45RO and prolyl-4-hydroxylase were Alexa Flour 488 and 647 (Molecular Probes, Eugene, OR), respectively. For CD34 and procollagen I detection; fixed tissue sections were dehydrated, treated with 1% trypsin, covered with blocking solution (PBS containing 3%BSA, 5% fat-free milk powder, and 1% goat serum) and incubated with primary antibody mouse anti-human CD34 (BD Biosciences, Pharmingen, Leiden, The Netherlands), rat anti-human procollagen I (Chemicon, Europe Ltd, Hampshire, United Kingdom and nucleic staining (Sytox Blue Nucleic Acid Stain, Molecular Probes, Eugene, OR). Sections were then wash with TBS+0.5% Triton X and blocked with 10% goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS. Secondary antibodies used for detection of CD34 and procollagen I were Alexa Flour 488 and 633 (Molecular Probes, Eugene, OR), respectively. Control experiments were performed with/without primary antibody or with/without secondary antibody. Control sections were included in all experiments to correct for background fluorescence. Isotype controls were used in combination and for each fluorochrome to decrease the risk of detecting unspecific staining. Fibrocytes were counted in a subepithelial zone 0–210 μm deep into the reticular zone and divided into area A, B and C (each area 70 μm deep). Results were expressed as the number of cells per 1 mm2. All sections with an intact basement membrane and a minimum requirement of intact tissue 70 μm into the reticular zone were counted. Sections were analyzed with Leica confocal-scanning equipment TCS SP2 II (Leica, Wetzlar, Germany).
Quantification of fibrocytes in cultured BALF fibroblasts
BALF fibroblasts (5000 cells) were cultured 48 h in 4 well chambers. Thereafter 200 cells were randomly choosed for cell counting for their different markers for fibrocytes differentiation.
Statistical analysis
Mean values ± standard errors of the mean (SEM) were calculated and Kruskall-Wallis was used for analyses of statistical significance (SPSS v7.0, Chicago, IL). All values of p < 0.05 were considered significant. Spearman coefficient of rank correlation was used to assess the degree of association between basement membrane thickness and number of fibrocytes.