SEPARATION OF ALVEOLAR MACROPHAGES AND DENDRITIC CELLS VIA AUTOFLUORESCENCE - PHENOTYPICAL AND FUNCTIONAL-CHARACTERIZATION

In a previous study we demonstrated that an increase of monocytes and dendritic cells (MDC) was found in the bronchoalveolar lavage fluid (BAL) after intratracheal instillation of a bacillus Calmette-Guerin suspension. In the current study, the bright autofluorescence of alveolar macrophages (AMs) was used to separate them efficiently from the MDC. Sorting of freshly isolated BAL cells resulted in a high-autofluorescent fraction, consisting predominantly of AMs, and a low-autofluorescent fraction containing the MDC, lymphocytes, and granulocytes. Thus, a clear separation between suppressive (AM) and stimulating (MDC) activity was obtained as shown in antigen-specific T cell responses. Flow cytometric parameters, density fractionation, and a series of ED monoclonal antibodies raised against rat macrophage antigens showed that both AMs and MDC were diverse populations. After overnight culture, more than 80% of an MDC population with a density range of 1.065-1.079 changed to a lower density (< 1.056) and morphologically developed into DCs with many processes. Concomitantly, monoclonal antibody ED1 expression changed from a granular pattern to a discrete juxtanuclear spot localization.