CELL-CYCLE EFFECTS AND INDUCTION OF APOPTOSIS CAUSED BY INFECTION OF HL-60 CELLS WITH HUMAN GRANULOCYTIC EHRLICHIOSIS PATHOGEN MEASURED BY FLOW AND LASER-SCANNING CYTOMETRY

Human granulocytic ehrlichiosis (HGE) is an occasionally severe and ev en fatal disease caused by an agent closely related to Ehrlichia equi and Ehrlichia phagocytophila, which is transmitted by ticks. Little is known about the pathogen itself, which only very recently has been is olated. The agent can be cultivated in vitro because it replicates in human promyelocytic leukemic HL-60 cells. Using multiparameter flow cy tometry and laser scanning cytometry (LSC) we have investigated change s in HL-60 cells following their infection with the pathogen, Its pres ence within the infected HL-60 cells was detected and its intracellula r level measured immunocytochemically using antibodies obtained from H GE-infected patients. The percentage of the infected cells measured by flow cytometry or LSC correlated well with the estimates by microscop y on the Giemsa-stained specimens, In the infected cultures, the cells had diminished levels of cyclins D3 and E as well as the cyclin depen dent kinase inhibitor p21(WAF1/CIP1) and were arrested predominantly i n G(0/1). The apoptosis-associated regulatory proteins were also affec ted by cell infection: expression of Bcl-2 was decreased in the infect ed cells whereas expression of Bar become more variable, with some cel ls showing higher levels of this protein. The infected cells developed numerous DNA strand breaks characteristic of apoptosis, The presence of the pathogen was also detected by LSC in cells from peripheral bloo d of the infected patients; after relocation and visual inspection ('' CompuSort'') the pathogen-positive cells were identified as leukocytes . This unique ability of LSC to detect, quantify, and visualize HGE in infected cells made this instrument particularly useful to measure th e degree of infection in peripheral blood of the patients and study ef fects of the infectious agent on the cell cycle and apoptosis of the h ost cells, (C) 1998 Wiley-Liss, Inc.