MACROPHAGE FUNCTIONAL-HETEROGENEITY INVIVO - MACROLOCAL AND MICROLOCAL MACROPHAGE ACTIVATION, IDENTIFIED BY DOUBLE-STAINING TISSUE-SECTIONS OF BCG GRANULOMAS FOR PAIRS OF ENZYMES

Abstract

BCG lesions were produced in rabbit skin and biopsies were performed at 7, 21 and 42 days, when they were developing, maximal in size and almost healed, respectively. Tissue sections were prepared and stained histochemically for several enzymes. The percentage of cells stained for a given enzyme and the distribution of such cells within lesions of various ages were determined. The 7-day BCG lesions contained few esterase- and .beta.-galactosidase-positive macrophages but 21-day lesions contained many, especially in the viable and nonviable tuberculous granulation tissue at the edge of the now prominent caseous necrotic center. Both 7- and 21-day lesions contained many acid phosphatase- and cathepsin-D-positive macrophages, which were numerous in the more peripheral parts of the lesion where little or no necrosis was present. Enzyme patterns in 42-day lesions resembled those in 21-day lesions. Although the role of each of these enzymes in the development and regression of the BCG lesion is unknown, this macrophage population is heterogeneous and macrophages carry out different functions in different parts of the lesion at different times. Histochemical techniques were developed to stain 2 enzymes in the same tissue section. The 1st stain usually contained a naphthol substrate and produced a red color; the 2nd stain contained an indoxyl substrate and produced a blue color. A cell staining with both was colored purple. The peroxidase-antiperoxidase immunocytochemical technique for cathepsin D (producing a red color) was also employed. Red esterase (hydrolyzing naphthol AS-D acetate) and .beta.-galactosidase, and red esterase and blue esterase (hydrolyzing 5-bromo-4-chloro-indoxyl acetate), probably the same enzyme, were usually present in the same macrophage. Each of the following enzyme pairs was usually present in a different macrophage: cathepsin D and .beta.-galactosidase; cathepsin D and blue esterase; acid phosphatase and .beta.-galactosidase; and acid phosphatase and blue esterase. Roughly 10% of the macrophages stained for 1 enzyme existed side by side with macrophages stained for a different enzyme. Local macrophage activation apparently is under 2 levels of control. The first, macrolocal control, determines the overall enzyme distribution in the lesion; the second, microlocal control, determines enzyme distribution on a cell-by-cell basis, i.e., how 2 neighboring macrophages can each be rich in a different enzyme.