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This study was designed to examine the cellular distribution of the angiotensin II type-1 (AT1) and type-2 (AT2) receptors in the normal human and pathological human lung. Riboprobes were prepared against specific portions of each receptor DNA and labelled with FITC for detection using an anti-FITC antibody in combination with the alkaline phosphatase-anti-alkaline phosphatase technique and new Fuchsin. These were used to detect the presence of receptor mRNA in the lung. Specific antibodies were used to detect receptor protein in cells by immunocytochemistry. Image analysis was used in order to semi-quantify receptor density. AT1 receptor mRNA and protein were localised on vascular smooth muscle cells, macrophages and in the stroma underlying the airways epithelium probably relating to underlying fibroblasts. The AT1 receptor protein was not expressed in the epithelium although there was a low level of mRNA. In contrast, AT2 receptor RNA and protein was observed in the epithelium, with strong staining on the bronchial epithelial cell brush border and also on many of the underlying mucous glands. The AT2 receptor was also present on some endothelial cells. These findings were supported by the presence of mRNA in each case. In patients with chronic obstructive pulmonary disease, there was a five- to sixfold increase in the ratio of AT1 to AT2 receptors in the regions of marked fibrosis surrounding the bronchioles. This correlated well with the reduced lung function as expressed by the forced expiratory volume.

1 The influence of the vascular endothelium on agonist-induced contractions and relaxations has been measured using intact segments of rat aorta. Contiguous rubbed segments were used as controls. 2 Angiotensin II, histamine, noradrenaline, U46619 and UK14304 contracted both rubbed and intact tissues. The threshold spasmogenic concentrations of these agonists were lower in rubbed tissues than in intact preparations. 3 The sensitivity and responsiveness of tissues to angiotensin II, histamine, noradrenaline and UK14304 were greater in rubbed than in intact tissues. 4 Acetylcholine and histamine relaxed the established spasms ofintact tissues but not those of rubbed preparations, These relaxant effects of acetylcholine were abolished by pre-incubation with haemoglobin. 5 In the presence of prazosin, noradrenaline or UK 14304 relaxed established contractions in intact tissues. These effects were antagonized by idazoxan or by pre-incubation with haemoglobin. 6 In intact preparations, idazoxan had no effect on the spasmogenic sensitivity and responsiveness to UK14304. 7 Pre-incubation with haemoglobin augmented the spasmogenic actions of noradrenaline, U46619 or UK 14304 in intact tissues, but had no effect on these responses in rubbed preparations. 8 Tissue concentrations of cyclic GMP were greater in intact than in rubbed tissues. A concentration of acetylcholine (10 tiM) evoking just maximal mechanical inhibition produced a significant increase in cyclic GMP concentration in intact preparations. However, no detectable changes in cyclic GMP concentration were produced by UK 14304 (10 !M) or by acetylcholine (30 nM), concentrations which were equi-effective in inhibiting mechanical activity. 9 In the presence of threshold spasmogenic concentrations of noradrenaline, the contractile effects of angiotensin II were augmented and became comparable to those observed in rubbed preparations. In the presence of greater concentrations of noradrenaline, angiotensin II always produced an additional contraction. 10 It is concluded that the presence of the vascular endothelium limits the spasmogenic action of a variety ofagonists. Although spasmogens like noradrenaline and UK 14304 can stimulate the release of endothelium-derived relaxing factor (EDRF) via M2-adrenoceptors, the inhibitory effects of EDRF largely result from the spontaneous release of this substance.

Bronchial biopsies have made possible the detailed study of the pathology of the airways of humans with respiratory disease. Much data has been accumulated on asthmatics or normal controls but much less is known about chronic bronchitics. The aim of this study was to characterize the cellular and cytokine pattern seen in chronic bronchitics and to compare these with control and asthmatic subjects. The patients were also characterized clinically. In this study, immunocytochemistry on cryostat sections from bronchial biopsies were used to determine the level of inflammatory cells and cells of the immune system as well as the pattern of cytokines. This study revealed a distinct cellular and cytokine pattern for each of the three different patient groups, although the diversity of the cytokines analysed was limited by the size of the biopsies. In the inflammatory infiltrate of patients with asthma, CD4+ T-cells and eosinophils were the most prominent cell types discerned. All of the expected cytokines such as IL-1, TNF-alpha, IL-4, IL-5 and IFN-gamma were found. In contrast, the emphasis in chronic bronchitic patients was quite different. The predominant cell types were macrophages, neutrophils, mast cells and CD8+ T-cells, but eosinophils were also abundant. In addition, IL-4 and TNF-alpha were the only cytokines present of those tested.

Two different receptors which bind angiotensin II specifically have been identified in humans and were designated angiotensin II type-1 receptor (AT1) and angiotensin II type-2 receptor (AT2). They only have 34% sequence homology and act through different signalling pathways. AT1 stimulation has been implicated in hypertrophy and hyperplasia in various tissues. In order to study the involvement of AT1 in tissues from controls (n=10) and patients with hyperplasia (n=33), ductal carcinoma in situ (DCIS) (n=23) and invasive carcinoma of the breast (n=25), we tested biopsies and breast-derived cell lines using immunocytochemistry, in situ hybridisation and cell proliferation techniques. The results show specific overexpression of AT1 receptor on the cytoplasmic membrane of cells of hyperplastic lesions with and without atypia and on DCIS of the breast. Evidence for growth stimulation is provided by in vitro experiments showing growth induction by angiotensin II of T47D cells which express the AT1 but not the AT2 receptor. The expression of AT1 on the cell membrane disappears in invasive breast cancer cells suggesting a regulatory pathway which is no longer needed in invasive carcinoma. The specific AT1 expression upregulation might well be an important step in the pathogenesis of hyperplasia of the breast, which is regarded as a precursor lesion for breast cancer.

SUMMARY Chemical fixation of cells has been used extensively in different fields of electron microscopy. The type of fixation can be selected to maximize retention of different cell constituents where morphology is of prime interest. For techniques such as immunocytochemistry and autoradiography, a compromise between morphology and retention of antigenicity or radiolabel has to be reached. By careful choice of fixative, buffer and other physical parameters, optimum results can be obtained for both animal and plant tissues. Emphasis has been put on more recent developments in technique.

Delivering antigen to the respiratory tract via professional antigen-presenting DCs sensitizes for a secondary response to inhaled antigen leading to airway inflammation. This model will prove very useful for studying the early events of sensitization to inhaled antigen using the respiratory route.

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