Rapid Turnover and High Production Rate of Myeloid Cells in Adult Rhesus Macaques with Compensations during Aging

Abstract

Neutrophils, basophils, and monocytes are continuously produced in bone marrow via myelopoiesis, circulate in blood, and are eventually removed from circulation to maintain homeostasis. To quantitate the kinetics of myeloid cell movement during homeostasis, we applied 5-bromo-2'-deoxyuridine pulse labeling in healthy rhesus macaques (Macaca mulatta) followed by hematology and flow cytometry analyses. Results were applied to a mathematical model, and the blood circulating half-life and daily production, respectively, of each cell type from macaques aged 5-10 y old were calculated for neutrophils (1.63 ± 0.16 d, 1.42 × 10⁹ cells/l/d), basophils (1.78 ± 0.30 d, 5.89 × 10⁶ cells/l/d), and CD14⁺CD16^- classical monocytes (1.01 ± 0.15 d, 3.09 × 10⁸ cells/l/d). Classical monocytes were released into the blood circulation as early as 1 d after dividing, whereas neutrophils remained in bone marrow 4-5 d before being released. Among granulocytes, neutrophils and basophils exhibited distinct kinetics in bone marrow maturation time and blood circulation. With increasing chronological age, there was a significant decrease in daily production of neutrophils and basophils, but the half-life of these granulocytes remained unchanged between 3 and 19 y of age. In contrast, daily production of classical monocytes remained stable through 19 y of age but exhibited a significant decline in half-life. These results demonstrated relatively short half-lives and continuous replenishment of neutrophils, basophils, and classical monocytes during homeostasis in adult rhesus macaques with compensations observed during increasing chronological age.

FIGURE 1. Phenotype analysis of neutrophils and basophils in peripheral blood of rhesus macaques

EDTA-treated blood samples were processed, stained with antibodies (Table I), and analyzed by flow cytometry as described in the Materials and Methods. (A) The gating strategies of myeloid-lineage cells were indicated for; (a) neutrophils = single cells, FSC/SSC^high/dim, HLA-DR⁻, CD3⁻, CD20⁻, CD123⁻ ; (b) basophils = single cells, FSC/SSC^high/dim, HLA-DR⁻, CD123⁺; and (c) classical monocytes = single cells, FSC/SSC^dim, HLA-DR⁺, CD14⁺CD16⁻. (B) Myeloperoxidase (MPO) expression was shown for neutrophils, basophils, classical monocytes, and lymphocytes gated from the above strategy.

FIGURE 2. Neutrophils, basophils, and monocytes exhibit distinct kinetics in blood during homeostasis

Single-bolus BrdU (60 mg/kg body weight) was administrated intravenously to eleven rhesus macaques aged 5 – 10 years old and EDTA-treated blood samples were collected 1, 2, 4, 7, and 14 days later for staining and flow cytometry analysis. Neutrophils (A), basophils (B), and classical monocytes (C) were gated based on the strategy described in Figure 1A and the percentages of BrdU-labeled cells were measured in each subset. Results from monocytes in panel C were previously published by Sugimoto et al. (8) and presented here for comparison with permission of the publisher.

FIGURE 3. Rapid turnover of neutrophils, basophils and classical monocytes, and massive production of neutrophils in rhesus macaques during homeostasis

(A) Turnover rates for neutrophils, basophils, and classical monocytes (k values as described in the Material and Methods section) were calculated from the best-fit curves to fit the mathematical model to the BrdU incorporation kinetics. (B) Half-life values for neutrophils, basophils, and classical monocytes were calculated as ln(2)/k. (C) Daily cell production numbers for neutrophils, basophils, and classical monocytes were calculated as absolute cell counts divided by the average circulating lifespans. Each data point represents results from one animal (n=11). All results were expressed as the mean ± SD for each cell population.

FIGURE 4. Gating strategy and BrdU kinetics of neutrophil- and basophil-committed progenitor cells in bone marrow

Bone marrow aspirates were collected, stained and analyzed 1, 4, 7, and 10 days after intravenous BrdU administration from 8 of the 11 animals assessed in Fig. 2. (A) Neutrophil-differentiating cells were gated as Singlet/CD45⁺/FSC-SSC^high/CD3⁻/CD20⁻/CD11b⁺/HLA-DR⁻. (C) Basophil-differentiating cells were gated as singlet/FSC-SSC^high/CD3⁻/CD20⁻/CD123^bright/HLA-DR⁻. The percent of BrdU-labeled (B) neutrophil- and (D) basophil-committed cells in bone marrow was low at day 1, increased at day 4, and then was declining by days 7 - 10.

FIGURE 5. Chronological aging correlated with lower production of neutrophils and basophils, and a shorter half-life of classical monocytes

EDTA-treated blood samples from rhesus macaques were collected and stained (group 2; Table II) 1, 4, 7, 10, and 14 days after iv BrdU administration. The BrdU⁺ cell fraction, half-life, and daily production were measured and calculated respectively for neutrophils (A, D, H), basophils (B, E, G), and classical monocytes (C, F, I). Blood kinetics were expressed as the mean ± SD of the BrdU⁺ cell fraction for each age group. Nonparametric Spearman correlation tests were used to compare half-life and daily production of cell types against age of the animals. P < 0.05 was considered statistically significant.