The role of Rac2 in regulating neutrophil production in the bone marrow and circulating neutrophil counts

Abstract

Circulating neutrophils are persistently higher in mice deficient in the small GTPase Rac2 than in wild-type (WT) mice. Therefore, we examined the mechanisms through which the small GTPase Rac2 regulates neutrophil production and release. Lethally irradiated WT mice reconstituted with a 50:50 mixture of WT and Rac2(-/-) fetal liver cells were protected from neutrophilia, suggesting that neutrophilia is primarily because of extrinsic defects that can be corrected by WT leukocytes. However, the differential counts and numbers of leukocyte subtypes differed between Rac2(-/-) and WT cells, suggesting that Rac2 modulates leukocyte lineage distribution. Kinetic studies suggest Rac2 modulates the release of neutrophils into the circulation and does not prolong their circulating half life. The percentage of bone marrow cells that expressed the neutrophil marker Gr-1 in lethally irradiated WT or Rac2(-/-) recipients of Rac2(-/-) stem cells was greater than in recipients of WT stem cells; however, circulating neutrophil counts were higher only in Rac2(-/-) recipients of Rac2(-/-) stem cells. Rac2 mRNA was expressed in the bone marrow of WT recipients of Rac2(-/-) stem cells and in human mesenchymal stem cells. The data presented here suggest that Rac2 in hematopoietic cells regulates leukocyte lineage distribution and Rac2 in nonhematopoietic cells might contribute to regulating circulating neutrophil counts.

Figure 1

The proportion of Gr-1⁺ cells that incorporated BrdU in the BM. BM cells flushed from femurs of WT and Rac2^−/− animals (Rac2^−/−) were stained with antibodies to Gr-1 and BrdU at the indicated times after BrdU administration. At 24 and 48 hours after BrdU administration, a significantly greater proportion of Gr-1⁺ cells in the BM of WT mice are BrdU⁺ compared to Rac2^−/− animals. Data are expressed as mean ± SEM (n = 4 to 10 animals per time point). *P < 0.05 compared to WT at this time point (Mann-Whitney U-test).

Figure 2

The appearance of BrdU-labeled neutrophils in the circulation of WT and Rac2^−/− animals. Circulating leukocytes from WT and Rac2^−/− animals were stained with antibodies to BrdU and Gr-1 at the indicated time points after receiving BrdU. A: The number of BrdU-labeled circulating neutrophils in WT and Rac2^−/− animals. B: The proportion of circulating neutrophils that incorporated BrdU in WT and Rac2^−/− animals. BrdU-labeled neutrophils were categorized according to the intensity of staining with fluorescein isothiocyanate-conjugated anti-BrdU (see Materials and Methods). C and D: The number and the proportion of bright-staining BrdU⁺ circulating neutrophils, respectively. Data are expressed as mean ± SEM (n = 4 to 10 at each time point). *P < 0.05 compared to WT animals (Mann-Whitney U-test).

Figure 3

A: Circulating neutrophil counts in healthy age- and sex-matched WT and Rac2^−/− mice. Neutrophil counts were determined using a hemocytometer as described in the Materials and Methods. Data are expressed as mean ± SEM (n = 5). *P < 0.05 versus WT (t-test). B: G-CSF in the circulation of WT and Rac2^−/− mice measured by enzyme-linked immunosorbent assay (R&D Systems). Plasma was obtained from healthy 8-week-old female mice, and the amount of G-CSF measured according to the manufacturer’s instructions. Data are expressed as mean ± SEM (n = 5). *P < 0.05 versus WT (t-test). Although Pearson’s product moment statistic may not be appropriate to test for correlation between these discrete sets of data, the number of circulating neutrophils did correlate with plasma G-CSF in WT and Rac2-deficient mice (r² = 0.67, P < 0.001).

Figure 4

The expression of Rac2 message in chimeric and control animals at least 12 weeks after 7 Gy irradiation and reconstitution. Total RNA was obtained from BM cells and the amount of Rac2 message in was determined using quantitative real-time RT-PCR. The amount of Rac2 message in the samples was quantified by comparing with PCR-amplified Rac2 cDNA standards. Data expressed as mean ± SEM (n = 4 to 5 mice per group). *P < 0.05 versus recipients of Rac2^−/− stem cells (analysis of variance and Scheffé’s post hoc test).

Figure 5

Expression of Rac2 message in cultured MSCs from normal human donors. Total RNA was isolated from cultured human MSCs, and identical amounts of starting total RNA were used in the RT reaction for each sample. The amount of Rac2 message was then quantified using real-time quantitative PCR by comparing with purified PCR-amplified standards, as described in the Materials and Methods. The amount of Rac2 message is expressed as ng of cDNA per μl of reaction volume. Data are shown as the mean ± SEM of three separate runs.

Figure 6

The proportion of WT and Rac2^−/− leukocytes in the blood and BM that are Gr-1⁺ in mice reconstituted with a 50:50 mixture of WT and Rac2^−/− fetal liver cells. Blood and BM cells were obtained at least 20 weeks after 12 Gy irradiation and reconstitution with an equal mixture of WT and Rac2^−/− stem cells. The samples were stained with antibodies to CD45.1 to identify WT leukocytes and CD45.2 to identify Rac2^−/−leukocytes and Gr-1. The proportion of WT (CD45.1⁺) and Rac2^−/− (CD45.2⁺) leukocytes that expressed Gr-1 in the blood (left) and BM (right) was calculated. Data are expressed as mean ± SEM (n = 6). In each of the mice studied, a greater proportion of Rac2^−/− leukocytes were myeloid cells (Gr-1⁺) compared with WT leukocytes. *P < 0.05 (paired t-test).