ACTIVATION OF PERIPHERAL-BLOOD MONONUCLEAR-CELLS IN BRONCHOALVEOLAR LAVAGE FLUID FROM PATIENTS WITH SARCOIDOSIS - VISUALIZATION OF SINGLE-CELL ACTIVATION PRODUCTS

Background - Interstitial lung diseases are characterised by the recru itment of mononuclear cells to disease sites where maturation occurs a nd activation products, including lysozyme (LZM), are released. Analys is of in vitro cell culture supernatants for activation products masks the functional heterogeneity of cell populations. It is therefore nec essary to examine the secretion of activation products by single cells to assess whether the activation of newly recruited mononuclear phago cytes at the sites of disease in the lung is uniform and controlled by the local microenvironment. Methods - The reverse haemolytic plaque a ssay was used to evaluate, at a single cell level, the ability of bron choalveolar lavage (BAL) fluid from seven patients with sarcoidosis to activate Ficoll-Hypaque-separated peripheral blood mononuclear cells by comparison with BAL fluid from six normal volunteers and nine patie nts with systemic sclerosis. Monolayers of peripheral blood mononuclea r cells and sheep red blood cells were cultured either alone or in the presence of 20% (v/v) BAL fluid with a polyclonal anti-LZM antibody. LZM/anti-LZM complexes bound to red blood cells surrounding the secret ing cells were disclosed following complement lysis of red blood cells and quantification of plaque dimensions using microscopy and image an alysis. Results - Bronchoalveolar lavage fluid from all the patients w ith sarcoidosis increased LZM secretion by peripheral blood mononuclea r cells compared with unstimulated mononuclear cells. By contrast, BAL fluid from the other individuals had no effect on LZM secretion. Conc lusions - Single cells activated by BAL fluid can be evaluated by the reverse haemolytic plaque assay. BAL fluid from patients with sarcoido sis, but not from patients with systemic sclerosis or normal individua ls, contains components capable of activating mononuclear phagocytes t o secrete lysozyme.