| Type I and III procollagen propeptides in sarcoidosis, fibrosing alveolitis and asbestos-related lung diseases | ||
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The chest radiography included posteroanterior and lateral views. A physician specialized in respiratory medicine interpreted them first. The radiographs were then interpreted retrospectively and blindly by an experienced thoracic radiologist.
The pulmonary function tests included forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), which were measured with a flow-volume spirometer. Diffusion capacity (DLCO) and the specific diffusion coefficient (DLCO/VA) were analysed by the single-breath technique.
Fiberoptic bronchoscopy for sampling BALF was performed under local anaesthesia with lignocain. The patients were premedicated with diazepam and atropine. A bronchofiberoscope was wedged into the right middle lobe or the left lingula. Saline solution was then installed in 10 aliquots of 20 ml. After gentle aspiration, the recovered fluid was collected in a plastic bottle and kept on ice. After centrifugation (400xg for 15 min), 30 ml of the cell-free supernatant was stored for later assays.
The serum angiotensin-converting enzyme was analysed with a method modified for the Kone Specific analyser (Kone Oy, Espoo, Finland) (Harjanne 1984). Interleukin-2-receptor was analysed with enzyme-linked immunosorbent assay (ELISA) (DAKO A/S, DK 2600 Glostrup, Denmark). Serum and BALF-urea and albumin and serum alanine aminotransferase (ALAT) were assayed with routine laboratory methods.
The total number of cells in BALF was counted in a Bürger haemocytometer, and their viability was assessed by trypan blue exclusion. 1 ml of lavage fluid was fixed in an equal volume of 95% ethanol, and Millipore filter preparations were made with Papanicolaou staining. Cytocentrifuge preparations (700 rpm, 5 min; Cytospin 2, Shandon Instruments, Astmoor, UK) of unfixed cells (roughly 100.000 cells/slide) were air-dried overnight at room temperature and stained with May-Grünwald-Giemsa. For differential counts, 200 cells were counted on both filter and cytocentrifuge preparations.
The amount of epithelial lining fluid (ELF) was calculated with the urea method (Rennard et al. 1986).
The number of asbestos bodies in BALF was determined from Millipore filter and cytocentrifuge (Cytospin 2, Shandon Instruments, Astmoor, UK) preparations. Two or three filter preparations containing 5 ml of lavage fluid each and four or five cytocentrifuge preparations containing 0.4 ml of lavage fluid each were prepared as described above. Both the Millipore filter and the cytocentrifuge preparations were stained with Pearl’s iron stain. The filter preparations were mounted on glass slides, and both the filter and the cytocentrifuge preparations were covered with coverslips. Asbestos bodies were identified on the basis of the previously described criteria (Churg & Warnock 1981) using a light microscope with a 20x objective. The number of asbestos bodies was expressed as per ml of BALF.
Serum procollagen I carboxyterminal propeptide (Melkko et al. 1990) and procollagen III aminoterminal propeptide (Risteli et al. 1988) were assayed by commercial radioimmunoassay kits (Orion Diagnostica Oy, Oulunsalo, Finland) employing human antigen and specific polyclonal antibodies.
For assaying PIIINP and PICP in bronchoalveolar lavage fluid by radioimmunoassay, the original methods for serum were modified in such a way that no concentration or further processing of the sample was necessary. The BALF samples used were bigger (1 ml) than the samples used in the serum methods (200 µl with PIIINP and 100 µl with PICP), and the tracer and antiserum amounts were, respectively, smaller but more concentrated compared to the original method. Human antigens were employed to induce polyclonal anti-PIIINP and anti-PICP antibodies. In the BALF-PIIINP assay, 1 ml of BALF or appropriate standard, adjusted to 0.1M phosphate buffer, was incubated with 200 µl of 125I-PIIINP tracer solution diluted 1:1000 with phosphate-buffered saline containing 0.04 % Tween -20 (PBS/Tween) and 100 µl of antiserum (diluted 1:1000 with PBS/Tween) at 37 °C. After 2 h incubation, 1.5 ml of secondary antibody (the original solid phase reagent concentrated x2) was added, and the tubes were further incubated at room temperature for 30 min and centrifuged at 3400 x g at 4 °C for 15 min, after which the radioactivity of the precipitates was counted. The standard curves were calculated using an automatic gamma counter (Clinigamma 1272, Wallac, Turku, Finland). The BALF-PICP assay was performed similarly: 200 µl of the 125I-PICP tracer solution (dilution 1:2500) and 150 µl of the antiserum (dilution 1:1000) were incubated with the samples, after which the immunocomplex was precipitated with 1 ml of secondary antibody (the original solid concentrated x2). The results are expressed either as µg of propeptide per liter of recovered BALF or as µg of propeptide per liter of ELF. The sensitivities of the original methods are 0.2 g/l with 200 l serum sample for PIIINP and 1.2 g/l with 100 l serum sample for PICP. The intra- and interassay variations of the original methods for PIIINP are 4.3% and 5.3% and those for PICP 3.2% and 6.6 %, respectively.
Open lung biopsies were taken from different parts of the left or right lung. The biopsy material was fixed in 10% formalin under vacuum in order to expand the tissue and to remove air bubbles (Wagenvoort 1980) or perfused by injecting fixative into bronchioles, using a small syringe (Dail & Hammar 1994). The specimens were then dehydrated and embedded in paraffin. 4 µm sections were stained with hematoxylin-eosin, Giemsa, Verhoeff, van Gieson, Pearl’s iron, periodic acid-Schiff alcian blue and periodic acid-Schiff stains. Uninvolved peripheral lung tissue was obtained from five patients operated on for pulmonary malignancy and used as controls.
Primary human polyclonal antibodies for procollagen I aminoterminal propeptide (Melkko et al. 1996) and procollagen III aminoterminal propeptide (Risteli et al. 1988) were used. For immunohistochemistry, 4 µm serial sections were deparaffinized and pre-treated with 0.4% pepsin for 30 minutes at 37°C. Endogenous peroxidase activity was blocked by incubating the slides in 0.3% H2O2 in absolute methanol for 30 minutes. The tissue sections were incubated with 2% goat serum for 10 minutes. Primary antibodies for PINP and PIIINP were used in concentrations of 1:100 and 1:50, respectively. The sections were incubated with primary antibody at 4 °C overnight, followed by a biotinylated goat secondary antibody (at dilution of 1:300 for 10 minutes) and the avidin-biotin-peroxidase complex (both from Dakopatts, Glostrup, Denmark). The colour was developed with diaminobenzidine. The sections were counterstained with a light hematoxylin stain and mounted with Eukitt (Kindler, Frieburg, Germany). The negative control stain consisted of substituted phosphate-buffered saline (PBS) at pH 7.2.