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

Abstract

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 immunocytochemically 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. Cytometry 33:47–55, 1998. © 1998 Wiley‐Liss, Inc.

Gene expression profiling of human promyelocytic cells in response to infection with Anaplasma phagocytophilum

“…Although the role of karyopherin in A. phagocytophilum ‐induced apoptosis is speculative, its inhibition may result in a negative effect to cell growth. Bcl‐2 and Bcl‐2‐like proteins, downregulated in infected cells, are anti‐apoptotic factors whose expressions are reduced in HL‐60 during A. phagocytophilum infection (Bedner et al ., 1998). Ceramide, linked to cells undergoing apoptosis and shown previously to be upregulated in infected HL‐60 cells, was also upregulated by microarray analysis (DiPietrantonio et al ., 1998; 2000).…”

Laser Scanning Cytometry: Principles and Applications—An Update

“…In another study, eosinophils were identified by LSC as “false positive” apoptotic cells due to their nonspecific labeling with fluorescein-conjugated reagents (63). LSC was also helpful in distinguishing apoptotic cells from cells infected by Human Granulocytic Erlichiosis (64). Based on these observations, as well as other findings, it was concluded that LSC is the instrument of choice for the analysis of apoptosis (13, 65).…”

High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry: A potential source of error in analysis of blood samples utilizing fluorescein‐conjugated reagents in flow cytometry

“…LSC is a microscope-based cytometer that combines advantages of flow and image cytometry. One of the features of LSC is that it allows one to relocate the cells and correlate morphology with the fluorescence measurements on a cell-by-cell basis, e.g., after restaining with other fluorescent or absorption dyes (3,(12)(13)(14)(15).…”