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 even 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 isolated. The agent can be cultivated in vitro because it replicates in human promyelocytic leukemic HL-60 cells. Using multiparameter flow cytometry and laser scanning cytometry (LSC) we have investigated changes in HL-60 cells following their infection with the pathogen. Its presence within the infected HL-60 cells was detected and its intracellular level measured inmmunocytochemically using antibodies obtained from HGE-infected patients. The percentage of the infected cells measured by flow cytometry or LSC correlated well with the estimates by microscopy on the Giemsa-stained specimens. In the infected cultures, the cells had diminished levels of cyclins D3 and E as well as the cyclin dependent kinase inhibitor p21WAF1/CIP1 and were arrested predominantly in G0/1. The apoptosis-associated regulatory proteins were also affected by cell infection: expression of Bcl-2 was decreased in the infected cells whereas expression of Bax become more variable, with some cells 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 blood 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 the degree of infection in peripheral blood of the patients and study effects of the infectious agent on the cell cycle and apoptosis of the host cells.
Bedner
E
E
The Cancer Research Institute, New York Medical College, New York 10523, USA.
Burfeind
P
P
Hsieh
T C
TC
Wu
J M
JM
Aguero-Rosenfeld
M E
ME
Melamed
M R
MR
Horowitz
H W
HW
Wormser
G P
GP
Darzynkiewicz
Z
Z
D017209:Apoptosis; D002453:Cell Cycle; D004536:Ehrlichia / Q000502:physiology*; D016873:Ehrlichiosis / Q000469:parasitology; D005434:Flow Cytometry / Q000379:methods*; D006098:Granulocytes; D018922:HL-60 Cells; D006801:Humans; D007834:Lasers