Quantitative analysis of apoptotic cell death in granulomatous inflammation induced by intravenous challenge with Cryptococcus neoformans and bacillus Calmette-Guerin vaccine

Apoptotic cell death of macrophage has become recognized as a significant mechanism responsible for the resolution of inflammation. The purpose of this study was to examine how the apoptotic cell death involves the formation and resolution of granulomas in rats intravenously inoculated with Cryptococcus neoformans (Cr. neoformans) and Mycobacterium bovis-derived bacillus Calmette-Guerin (BCG) vaccine. The number and size of granulomas in the livers obtained on days 5, 10, 15, 20 and 25 after inoculation were examined by morphometric image analysis, as well as the occurrence of apoptotic cell death quantitatively analyzed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) procedure on tissue sections. in both groups the number and size of granulomas were maximized on day 10, then the granulomas were almost resolved until day 25 when the inoculated Cr. neoformans and BCG almost disappeared. From the induction to the resolving stages of granulomatous inflammation, TUNEL-positive cells constantly appeared in granulomas, and the highest frequency of apoptotic cells in granulomas was observed in the earlier stage of granuloma formation. These results indicate that the maintenance and resolution of infectious granulomas are regulated by the balance between the influx of newly recruited macrophages and the apoptotic elimination of granuloma macrophages. The apoptosis of granuloma macrophages actively involves the cellular turnover in both granuloma formation and resolution.