Human Cytomegalovirus Inhibits Granulopoiesis Via Direct Infection and Inducing Apoptosis.

Human cytomegalovirus (HCMV) infection can cause delayed leukocyte recovery after bone marrow transplantation and often associated with suppression of granulocyte/macrophage progenitor. Leukocyte lineage may be one of the major sites of HCMV infection. However, whether HCMV can interfere with CFU-GM formation and induce apoptosis in granulocyte/macrophage progenitor have not been well investigated. Human bone marrow mononuclear cells (Ficoll), promyelocyte cell line HL-60 and HCMV AD169 strain were co-cultured. Each bone marrow specimen was HCMV DNA and HCMV IgM negative by PCR and ELISA test. Our results showed that HCMV significantly inhibited the formation of CFU-GM as shown in two different concentrations of viral infection groups: 139.26 ± 5.42 (2×10⁵pfu/ml), and 124.19 ± 8.82 (2×10⁶pfu/ml) (colonies/2×10⁵cells/ml, n=26). These were significant differences compared with the blank control group (P<0.01, P<0.05) respectively. HCMV also significantly inhibited the growth of HL-60 cells in three different concentrations of viral infection groups (10¹pfu/ml, 10²pfu/ml, 10³pfu/ml). After incubation for 7 days, viability cells in control group and each infection groups (n=20) were 96%, 83%, 73%, and 64%, showing that HCMV infection decreased the viability of HL-60 cells in a dose-dependent manner. The apoptotic effect was also investigated by Annexin V assay using flow cytometry. The percentage of Annexin V -positive cells were 19.60 ± 1.63 (in group of 10¹pfu/ml) (P<0.05), 28.70 ± 2.61 (10²pfu/ml) (P<0.05), and 36.3 ± 2.57 (10³pfu/ml) (P<0.01), compared with control group 3.96 ± 1.75 (n=8). The apoptotic cells were further confirmed by morphologic observation and DNA ladder formation. Furthermore, HCMV DNA was positive in HL-60 cells and cells of CFU-GM measured by PCR and IS-PCR analysis respectively. These were negative in blank and mock control groups. Our data demonstrated that: (1) HCMV AD169 strain inhibited the growth of HL-60 and CFU-GM formation in a dose-dependent manner; (2) HCMV transcription occurred in HL-60 and CFU-GM cells; and (3) This virus induced apoptosis in HL-60 cells. HCMV may have an inhibiting effect on granulopoiesis via direct infection and inducing apoptosis on myeloid progenitors.