Reduced cell turnover in bovine leukemia virus-infected, persistently lymphocytotic cattle

Abstract

Although nucleotide analogs like bromodeoxyuridine have been extensively used to estimate cell proliferation in vivo, precise dynamic parameters are scarce essentially because of the lack of adequate mathematical models. Besides recent developments on T cell dynamics, the turnover rates of B lymphocytes are largely unknown particularly in the context of a virally induced pathological disorder. Here, we aim to resolve this issue by determining the rates of cell proliferation and death during the chronic stage of the bovine leukemia virus (BLV) infection, called bovine persistent lymphocytosis (PL). Our methodology is based on direct intravenous injection of bromodeoxyuridine in association with subsequent flow cytometry. By this in vivo approach, we show that the death rate of PL B lymphocytes is significantly reduced (average death rate, 0.057 day(-1) versus 0.156 day(-1) in the asymptomatic controls). Concomitantly, proliferation of the PL cells is also significantly restricted compared to the controls (average proliferation rate, 0.0046 day(-1) versus 0.0085 day(-1)). We conclude that bovine PL is characterized by a decreased cell turnover resulting both from a reduction of cell death and an overall impairment of proliferation. The cell dynamic parameters differ from those measured in sheep, an experimental model for BLV infection. Finally, cells expressing p24 major capsid protein ex vivo were not BrdU positive, suggesting an immune selection against proliferating virus-positive lymphocytes. Based on a comparative leukemia approach, these observations might help to understand cell dynamics during other lymphoproliferative disease such as chronic lymphocytic leukemia or human T-cell lymphotropic virus-induced adult T-cell leukemia in humans.

FIG. 1.

Primary PBMCs undergo apoptosis in short-term cultures. (A) Clinical status of cattle and cell phenotype of their PBMCs. Peripheral blood mononuclear cells were isolated from cows with PL (BKL-2, Stara) and asymptomatic (AS) BLV-infected cattle (BKL, Wysoka), as well as from three seronegative controls (uninfected: NI) (PBK, BK, 109322). The total leukocyte counts were determined and the number of lymphocytes was estimated after examination under the microscope. PBMCs were labeled with monoclonal antibodies directed against sIgM or CD5 and analyzed by flow cytometry. Numbers (± standard deviations), which were deduced from three independent experiments, represent the percentages of positive cells within the total PBMC population. (B) PBMCs were cultivated for 18 h in the absence (no chemical) or in the presence of PMA and ionomycin and labeled with anti-IgM monoclonal antibody 1H4 and a fluorescein isothiocyanate conjugate. After ethanol fixation, the cells were stained with propidium iodide and, after exclusion of the doublets, analyzed by two-color flow cytometry. Results from a representative experiment (10,000 events) are shown as dot plots (x axis: propidium iodide; y axis: B-lymphocyte labeling). Numbers within the plots represent the percentages of positively stained B cells in the PBMC population within each region.

FIG. 2.

Relative rates of apoptosis as well as the levels of proliferation depend on the culture conditions. (A) PBMCs were isolated from PL, asymptomatic (AS) or noninfected animals (NI) and cultivated ex vivo under spontaneous (no chemical) or optimized conditions (in the presence of PMA and ionomycin). Cells were then labeled with anti-IgM monoclonal antibody and stained with propidium iodide (as described for Fig. 1B). Mean percentages (± standard deviations) of B lymphocytes at different stages of the cell cycle (sub-G₁/apoptotic, G₀/G₁, S and G₂/M) were calculated from four independent experiments. (B) Graphic representation of the mean values and standard deviations from panel A. (C) Relative proportions of the B lymphocytes at various stages of the cell cycle under optimized conditions (in the presence of PMA and ionomycin). ***, highly statistically significant, P < 0.001 according to the Student t test.

FIG. 3.

Bromodeoxyuridine incorporates into B lymphocytes in vivo. Two PL (BKL-2, Stara) and aleukemic (BKL, Wysoka) BLV-infected cattle and three controls (PBK, BK, 109322) (PBK and 109322 are represented) were injected intravenously with 3 g of BrdU, and an aliquot of blood (1 ml) was collected 6 days later. After lysis of the red blood cells, B cells were labeled with biotinylated 1H4 monoclonal antibody and streptavidin-phycoerythrin (PE) conjugate. Then, the cells were stained with anti-BrdU fluorescein isothiocyanate antibody in the presence of DNase and analyzed by two-color flow cytometry (x axis = BrdU; y axis = B lymphocytes). Ten thousand cells (lymphocytes, monocytes, and granulocytes) were acquired and PBMCs were selected by the forward/side scatter gating method. The total numbers of B cells are indicated in the upper quadrants.

FIG. 4.

Altered kinetics of BrdU incorporation in PL cows. Blood samples from cows (see Fig. 3) were collected at different days after a single pulse of BrdU injection. The percentage of BrdU-positive cells within the total B-lymphocyte population was determined and the data corresponding to the measured incorporation rates were fitted to a mathematical model, yielding theoretical fit curves (see Materials and Methods). Figure shows the average data for three groups of cows within two experiments. PL, AS, and NI are, respectively, persistently lymphocytic (triangles), asymptomatic (squares), and noninfected (lozenges) animals.

FIG. 5.

Proliferation and death rates are reduced in PL cows. The minimal proliferation (panel A) and death (panel B) rates were estimated from fitting the model to the data deduced from two independent experiments. PL, AS, and NI are, respectively, persistently lymphocytic, asymptomatic, and noninfected animals. Statistical analysis reveals that the proliferation and death rates are significantly reduced in PL cows compared to uninfected and asymptomatic in both experiments: average proliferation rates of B cells in PL cows lower than in uninfected and asymptomatic, confidence level: 90% in experiment 1 (*), 95% in experiment 2 (**). Average death rates of labeled B cells in PL cows were lower than in uninfected and asymptomatic, confidence level: 95% in experiment 1 (**), 95% in experiment 2 (**) (two-tailed Student t test in each case).

FIG. 6.

Cell proliferation and viral expression appear mutually exclusive. Three days post-BrdU injection, PBMCs from noninfected (PBK, 109322), aleukemic (BKL, Wysoka), and PL (BKL-2, Stara) cows were isolated and cultivated for 18 h. The cells were then fixed and incubated with anti-p24 antibody 4′G9, which recognizes the viral capsid protein, and with a phycoerythrin-conjugated secondary antibody. Finally, cells were stained with anti-BrdU fluorescein isothiocyanate conjugate containing DNase and analyzed by flow cytometry. A representative experiment (out of three) is represented as dot plots (10,000 gated events). Numbers represent the percentages of positively stained cells in each quadrant.