Identification of macrophages in intimal thickening of rat carotid arteries by cytochemical localization of purine nucleoside phosphorylase.

Complete desquamation of the endothelium of the rat carotid artery by balloon catheter stripping resulted within 2 weeks in the formation of a large intimal thickening. After an enzyme cytochemical technique was applied to localize cytosolic purine nucleoside phosphorylase (PNP), light microscopical evaluation indicated that this intimal thickening in normocholesterolemic rats was composed of 5.8% to 11.8% (mean 8.8%) PNP-positive cells. At the electron microscopic level, all these PNP-positive cells were identified as macrophages by the absence of a basement membrane and plasmalemmal vesicles and by the occurrence of specific intracytoplasmic granules. The nearly nonreactive intimal cells were classified as modified smooth muscle cells. Additional evidence of the macrophage nature of the PNP-stained intimal cells was obtained by differential immunogold labeling of these cells with a monoclonal antibody against rat macrophages. Moreover, in hypercholesterolemic rats, only the cells stained for PNP transformed into foam cells (between 8.5% and 11.4% of all nucleated intimal cells; mean 9.6%). This study shows that PNP cytochemistry discriminates macrophages from modified smooth muscle cells in the rat carotid intimal thickening. It further suggests that the intimal thickening in normocholesterolemic rats originates not only from migration and proliferation of smooth muscle cells, but also from a considerable number of leukocyte-derived macrophages. Whether the latter cells are actively involved in the establishment of the intimal thickening as has been suggested in dietary hypercholesterolemia, remains to be verified.

SEM morphological studies of phagocytosis by rat macrophages and rabbit polymorphonuclear leukocytes.

Peritoneal rat macrophages and rabbit polymorphonuclear leukocytes were obtained after thioglycollate or glycogen stimulation. Optimal conditions for phagocytosis were determined by a recently developed quantitative fluorimetric assay. We studied by serial SEM micrographs macrophages and polymorphonuclear leukocytes incubated either in medium or in the presence of different types of phagocytic particles. We compared the morphological aspects of adsorption and phagocytic processes for opsonized microorganisms (Micrococcus lysodeikticus and Candida albicans) with these for inert beads of iron and metallic mercury. Without phagocytosis, in the presence of fresh homologous serum, we observed a progressive development of microvilli or lamellipodia in ruffles and by the end, hypertrophied ruffles appeared at one pole of the cell. We noted extremely well developed ruffles during phagocytosis of opsonized microorganisms. These were practically absent on the macrophages incubated with inert particles. The mean number of adsorbed particles is more elevated in the case of iron and metallic mercury beads than for microorganisms. The rate of ingestion of inert particles was considerably higher than for microorganisms even when they were opsonized. In conclusion, at all stages of the phagocytic process, we observe different morphological features of the macrophages depending on the nature of the phagocytosed particles.