E L S E V I E R Immunopharmacology 33 (1996) 317-320

Immunopharmacoloyy

Immunoreactive B 1 receptors in human transbronchial tissue

Raj Nadar a,*, Amanda Derrick b, Strini Naidoo a, Yugen Naidoo a, Fred Hess b, Kanti Bhoola a

a Department of Experimental and Clinical Pharmacology, University of Natal, Congella, South Africa b Molecular Pharmacology and Biochemistry, Merck Research Laboratories, Rahway, NJ 07065, South Africa

Keywords: Bradykinin; Sarcoidosis; Systemic sclerosis; Cell proliferation

1. Introduction

Sarcoidosis is a multisystem disorder character- ized histologically by the finding of non-caseating granulomas in affected tissues. The lungs are in- volved in more than 90% of cases. In the majority of patients the condition resolves spontaneously, but in certain individuals, it may progress to fibrosis. In interstitial lung disease associated with progressive systemic sclerosis the histological picture is often one of fibrosis, although a desquamative interstitial pneumonitis is often observed in the early phases. The link between inflammation and fibrosis is thought to be due to a cascade of chemical mediators such as kinins, interleukins and growth factors. The forma- tion of kinins within the lung and bronchial fluid may well be a factor in the induction of the cell proliferative responses seen. In a previous study, Poblete et al. (1993) demonstrated the presence of tissue kallikrein in seromucous glands of human bronchi.

Regoli et al. (1990) suggested that bradykinin and related kinin peptides probably mediate their effects via two distinct receptors, namely B 1 and B2. Gold-

* Corresponding author. Department of Pharmacology, P.O. Box 17039, Congella, South Africa, 4013.

stein et al. (1984) demonstrated that B1 receptor stimulation resulted in an increase in collagen syn- thesis, and cell multiplication in human fibroblasts from foetal lung. Stimulation of B1 receptors by kinins results in the release of interleukin-1 and tumour necrosis factor by macrophages (Bhoola et al., 1992). The recognised two types of bradykinin receptors present on the same cells may subserve different functions. The B2 receptors serving physio- logical functions whereas expression of B 1 receptors is possibly enhanced in pathological states or in response to inflammatory conditions. Recently, Menke et al. (1994) have cloned the B 1 receptor and produced an antibody to its C terminus. We report on a pilot study to determine whether B 1 receptors are present in diseased lung tissue.

2. Methods

Transbronchial biopsies were obtained from two patients with sarcoidosis and 3 patients with intersti- tial lung disease associated with progressive sys- temic sclerosis.

Bronchoscopy was performed using an Olympus BF 1T10 bronchofiberscope. Local anaesthesia was achieved by transtrachial administration of 10 ml of 0.5% bupivacaine. Patients were premedicated with 0.5 mg of atropine and then sedated with 5 mg of

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R. Nadar et al. / lmmunopharmacology 33 (1996) 317-320 319

intravenous midazolam. The bronchoscope was in- troduced orally, and airway anatomy inspected be- fore wedging the tip of the bronchoscope in a sub- segmental branch of the right middle lobe bronchus. A bronchoalveolar lavage was performed using 150 ml of warm physiological saline. Thereafter, trans- bronchial biopsies were taken.

2.1. Handling of transbronchial tissue

Sections were routinely stained with haematoxylin and eosin for light microscopy. The presence of fibrosis was confirmed using Masson's trichrome stain. Transbronchial specimens were also wax em- bedded after graded series of alcohol dehydration. Preparation for immunolabelling was performed as follows: 3 jxm sections were cut and mounted onto adhesive slides (starfrost, Radem), dewaxed rehy- drated in a graded series of alcohol and distilled water.

Initial blocking of non-specific receptor sites was achieved with 1% human IgG (1 mg/ml) for 30 min. Incubation with primary antibody (rabbit anti- human B 1 receptor IgG) was carried out in a humidi- fied chamber for 3 h. Cy3 (fluorochrome 552) la- belled sheep anti-rabbit IgG was used as the sec- ondary antibody. Immunofluorescence was visu- alised by confocal microscopy. (Leitz DM IRB con- focal microscope, Leica, Germany).

Controls: Lung tissue obtained from fresh corpses at a government mortuary where the cause of death was trauma, was used to perform controlled im- munostaining. Care was taken to obtain tissue from non-traumatized lungs. In addition, a blocking step was carried out by pre-incubating the primary anti- body directed to the C-terminal segment of the B 1 receptor with both the appropriate C-terminal pep- tide, as well as the inappropriate peptide, namely the N-terminus.

lung disease associated with progressive systemic sclerosis (Figs. 1 and 2). The most intense labelling was visualized predominantly in areas of fibrosis. Significant labelling was also demonstrated in the thickened interstitium, and in the basement mem- brane of capillaries and alveoli. There was no im- munolabelling in tissues obtained from the presumed healthy lungs, as well as with the preabsorbed spe- cific antibody. However, immunolabelling was ob- served when the antibody was preabsorbed with the inappropriate peptide (Fig. 3).

4. Discussion

This pilot study demonstrates the presence of B l receptors within the fibrous tissues of the lung and basement membrane of alveoli and capillaries. The absence of labelling in the normal tissue implies induction of B1 expression in pathological states of the lung. The strong immunofluorescence visualized in areas of fibrosis implies a greater density of B 1 receptor expression within fibrous tissue. This find- ing strengthens the demonstration of fibroblast pro- liferation reported by Goldstein et al. (1984). It is likely that the fibrous reaction observed in these tissues is perpetuated by kinins, amongst other possi- ble mediators. Whilst the exact role of kinins in inducing fibrosis remains to be elucidated further, it is our contention that it plays an important role in the pathogenesis of the interstitial lung disease seen in patients with sarcoidosis and progressive systemic sclerosis.

Acknowledgements

This study was supported by funds from the Medical Research Council of South Africa and the University of Natal Research fund.

3. Results

Immunofluorescence denoting the presence of B 1 receptors was visualized in all transbronchially biop- sled tissue. The strongest immunofluorescence was observed in sections from patients with interstitial

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