Early bone marrow hematopoietic defect in simian/human immunodeficiency virus C2/1-infected macaques and relevance to advance of disease

Abstract

To clarify hematological abnormalities following infection with human immunodeficiency virus (HIV), we examined the hematopoietic capability of bone marrow by using cynomolgus monkeys infected with pathogenic simian/human immunodeficiency virus (SHIV) strain C2/1, an animal model of HIV infection. The relationship between the progress of the infection and the CD4/CD8 ratio of T lymphocytes or the amount of SHIV C2/1 viral load in the peripheral blood was also investigated. A colony assay was performed to assess the hematopoietic capability of bone marrow stem cells during the early and advanced phases of the infection. Colonies of granulocytes-macrophages (GM) were examined by PCR for the presence of the SIVmac239 gag region to reveal direct viral infection. There was a remarkable decrease in the CFU-GM growth on days 1 and 3 postinoculation, followed by recovery on day 56. During the more advanced stage, the CFU-GM growth decreased again. There was minimal evidence of direct viral infection of pooled cultured CFU-GM despite the continuously low CD4/CD8 ratios. These results indicate that the decrease in colony formation by bone marrow stem cells is reversible and fluctuates with the advance of the disease. This decrease was not due to direct viral infection of CFU-GM. Our data may support the concept that, in the early phase, production of inhibitory factors or deficiency of a stimulatory cytokine is responsible for some of the bone marrow defects described in the SHIV C2/1 model.

FIG. 1.

Changes of CD4/CD8 ratio in monkeys inoculated with SHIV C2/1. All monkeys showed decreased CD4/CD8 ratios between day 14 and day 21 after inoculation. Monkey 4345 had a decrease in CD4/CD8 ratio in the first 24 h. Control monkeys showed only negligible declines in the first 24 h (preinoculation, 1.26 ± 0.400 [mean ± standard deviation]; 6 h, 1.24 ± 0.259; 24 h, 1.11 ± 0.323; n = 4).

FIG. 2.

Plasma viral load in the infected monkeys. Plasma viral RNA of four monkeys (200, 944, 520, and 844) was analyzed by PCR for the presence of the SIVmac239 gag region.

FIG. 3.

Colony assay on monkeys inoculated with SHIV C2/1. A period of days 1 through 3 after inoculation was defined as early stage, whereas days 56 through 238 were defined as advanced stage. P was <0.005 for CFU-GM, and P was <0.02 for CFU-E in comparison of early stage and advanced stage and of virus-inoculated monkeys and sham-inoculated controls at days 1 and 3. P values were calculated according to Kruskal-Wallis analysis. There was no statistically significant difference between sham-inoculated controls and non-sham-treated controls by Mann-Whitney analysis.

FIG. 4.

Morphology of colonies produced by BFU-E and CFU-GM. Photographs of colonies cultured in nitrocellulose medium are shown at an ×75 magnification by a microscope. The left column shows BFU-E, and the right column shows CFU-GM. The upper section shows colonies from uninfected monkeys, while the lower shows colonies from monkeys infected with SHIV C2/1.

FIG. 5.

Detection of SIVmac239 gag sequence in bone marrow colonies by PCR. Lanes: 1, a DNA ladder marker, HincII; 2, a negative control; 3, a positive control, a DNA sample from cell line M8166; 4, CFU-GM from monkey 4345 at the early stage; 5, CFU-GM from monkey 430 at the advanced stage; 6, CFU-GM from monkey 039 at the advanced stage (this monkey died of AIDS on day 238).