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Originally published online as doi:10.2353/ajpath.2008.071059 on June 26, 2008

Published online before print June 26, 2008
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(American Journal of Pathology. 2008;173:507-517.)
© 2008 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2008.071059

The Role of Rac2 in Regulating Neutrophil Production in the Bone Marrow and Circulating Neutrophil Counts

John C. Gomez*{dagger}, Jindrich Soltys*, Keiichi Okano*, Mary C. Dinauer{ddagger} and Claire M. Doerschuk*{dagger}

From the Department of Pediatrics,* Division of Integrative Biology, Rainbow Babies and Children’s Hospital and Case Western Reserve University, and the Department of Pathology,{dagger} Case Western Reserve University, Cleveland, Ohio; and the Cancer Research Institute,{ddagger} Indiana University School of Medicine, Indianapolis, Indiana


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
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.

The small GTPase Rac2 is a member of the Rho family of Ras-like small GTPases.1 In addition to Rac2, the Rac subfamily includes the ubiquitously expressed Rac1, which shares greater than 92% amino acid identity with Rac2. Both Rac1 and Rac2 are highly homologous to Rac3, whose expression has been reported in a variety of tissues including brain, heart, placenta, and pancreas.2 In contrast, Rac2 expression has been reported to be limited to hematopoietic cells.

Rac2 regulates a wide variety of functions, including cytoskeletal organization and rearrangements, superoxide production, chemotaxis, phagocytosis, transcription, and cell growth and proliferation.1 A patient bearing a dominant-negative allele of the Rac2 gene showed a mild granulocytosis, which was primarily attributable to an increase in circulating neutrophils and immature band forms, and had recurrent life-threatening infections and impaired wound healing.3-5 Neutrophils derived from this patient showed impaired chemotaxis in response to interleukin (IL-8) and fMLP and reduced superoxide production with fMLP stimulation.

Rac2–/– mice have circulating neutrophil counts that are several times those seen in wild-type (WT) animals.6 The bone marrow (BM) from Rac2–/– mice exhibits a slight increase in mature granulopoiesis.6 Neutrophils obtained from Rac2–/– animals demonstrate impaired F-actin rearrangements, lamellipodia formation, and chemotaxis in response to inflammatory mediators that signal through G protein-coupled receptors, although F-actin rearrangements in response to tyrosine kinase-coupled growth factors are not altered.6 Rac2–/– neutrophils also show decreased NADPH oxidase activity in response to fMLP, phorbol esters, and IgG-opsonized particles but not opsonized zymosan.6,7 Rac2–/– mice demonstrate decreased inflammatory responses and increased mortality in invasive aspergillosis compared to WT mice.6

Neutrophilia in Rac2 deficiency might be attributable to the inability of mutant leukocytes to defend the host from microbial pathogens, resulting in chronic infections that stimulate neutrophil production in the BM. However, the ability of Rac2 to control a broad range of processes in hematopoietic cells suggests that Rac2 may directly regulate neutrophil production and release from the BM. Interestingly, Rac2–/– mice are reported to have higher numbers of circulating hematopoietic stem cells/progenitors (HSC/Ps) at baseline and after G-CSF treatment compared to WT littermates despite having similar numbers of HSC/Ps in the BM,8 indicating that Rac2 may be important in the retention and mobilization of these cells from the BM. Rac2 has been reported to control apoptosis in HSC/Ps, in addition to its role in controlling migration and adhesion.9 Thus, Rac2 can regulate the number of hematopoietic progenitors in a cell-intrinsic manner, which is distinct from extrinsic mechanisms because of defects in host defense.

To determine the role of Rac2 in neutrophil production and release, neutrophil kinetics was compared in healthy WT and Rac2–/– mice. Leukocyte counts and proliferation of BM cells were studied in WT and Rac2–/– mice reconstituted with WT or Rac2–/– stem cells. Finally, the ability of Rac2–/– stem cells to reconstitute hematopoiesis was examined in lethally irradiated mice reconstituted with a mixture of WT and Rac2–/– stem cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals

Rac2–/– mice were generated by homologous recombination6 and backcrossed into C57BL/6J mice for more than 12 generations. CD18-deficient mice were generated as described and backcrossed into C57BL/6J mice for greater than 10 generations.10 The mutant mice are each completely null in the expression of the targeted molecule. WT C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). For BM transplant experiments, WT mice bearing the CD45.1 allele were used. All mice were housed in microisolator cages under specific pathogen-free conditions in a barrier facility and were given autoclaved food and water.

Neutrophil Kinetics

Neutrophil kinetics was examined in uninfected WT and Rac2–/– mice that were 6 to 10 weeks old. The animals received the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) to label dividing cells in the BM (1 mg per mouse intraperitoneally). BrdU (Sigma-Aldrich, St. Louis, MO) is incorporated in place of thymidine during DNA synthesis in dividing cells. Animals were sacrificed by halothane overdose at 12, 24, 48, 60, 72, 96, 120, 144, or 168 hours after BrdU injection. Blood was obtained from the inferior vena cava, and femurs were dissected. The contents of the dissected femur were flushed with cold phosphate-buffered saline containing 2 mmol/L ethylenediaminetetraacetic acid. Circulating leukocyte counts were determined using a hemocytometer and differential counts were determined using a blood smears stained with Hema3 Protocol (Fisher, Middletown, VA).

Blood and BM samples were stained with phycoerythrin-labeled anti-Gr-1 (BD Pharmingen, San Diego, CA) to identify neutrophils and fluorescein isothiocyanate-labeled antibody to BrdU (BD Pharmingen), according to a previously described protocol.11 The presence of BrdU+ neutrophils in the circulation and BrdU+ Gr-1+ cells in the BM was determined using a BD FACScan flow cytometer (BD Biosciences, San Jose, CA). Flow cytometry data were analyzed using BD CellQuest (BD Biosciences).

In the BM, the expression of Gr-1 is directly related to increasing granulocyte differentiation.12 High Gr-1 expression in the circulation is limited to neutrophils. In these studies, leukocytes were gated according to their forward and side-scatter characteristics. High surface expression of Gr-1 was used to identify neutrophils within the leukocyte gate. The percentage of Gr-1-expressing cells that stained positively for BrdU was determined for each blood and BM sample.

Up to 24 hours after BrdU administration, almost all BrdU+ cells stained brightly with the anti-BrdU antibody. BrdU bright neutrophils were selected as the subset of BrdU-labeled neutrophils that showed equal or greater staining intensity compared to BrdU+ cells at 24 hours after BrdU administration. The number of BrdU+ circulating neutrophils per ml of blood was calculated by multiplying the percentage of BrdU+ Gr-1+ circulating cells by the circulating neutrophil counts.

Measurement of Circulating G-CSF Levels and Lung IL-17 mRNA Expression

Age- and sex-matched WT, CD18-deficient mice, and Rac2–/– mice were sacrificed by isoflurane overdose. Blood samples were obtained from the inferior vena cava using a heparinized syringe. Plasma G-CSF levels were determined by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Total RNA was obtained from dissected lungs using Trizol (Invitrogen, Carlsbad, CA). RNA was reverse-transcribed using oligo (dT) and random hexamer primers, and Superscript III reverse transcriptase (Invitrogen). The amount of specific message was quantitated using real-time quantitative polymerase chain reaction (PCR) (ABI Prism 7000 sequence detector systems; Applied Biosystems, Foster City, CA). Quantitative PCR was performed using SYBR Green PCR master mix (Applied Biosystems). The PCR was at 10 minutes at 95°C, followed by15 seconds at 95°C and 60 seconds at 60°C for 40 cycles. The primer sequences were as follows: IL-17 forward: 5'-GTTCCACGTCACCCTGGAC-3', IL-17 reverse: 5'-CTTTCCCTCCGCATTGACAC-3'; 28S mt rRNA forward: 5'-TGTGGATGGCGAGAAATACCA-3', 28S mt rRNA reverse: 5'-GCATCAGCCTCCAGTATAGTTGT-3'. Fluorescence detection was reported at the end of each cycle. The quantity of message in each sample was correlated to the cycle at which fluorescence above baseline signal is first detected (CT). Each reaction was performed in triplicate, and the mean CT determined. The amount of IL-17 mRNA was then normalized to the expression of mitochondrial 28S ribosomal RNA in each sample and expressed as the IL-17 threshold cycle normalized by the amount of the endogenous housekeeping gene ({Delta}CT), as recommended by the instrument manufacturer (Applied Biosystems). Each unit difference in the corrected CT is approximately a twofold difference in the amount of starting IL-17 template. The relative expression of IL-17 message was calculated after setting the mean IL-17 expression in WT lungs as 1.

Hematopoietic Reconstitution

Livers from viable 13- to 14-day WT and Rac2–/– fetuses were excised and homogenized. Suspensions containing fetal liver cells (5 to 10 x 106/ml) were prepared. Recipient WT or Rac2–/– mice given trimethoprim-sulfamethoxazole in their drinking water for 1 week were lethally irradiated with 7, 8, or 12 Gy radiation from a 137cesium source. The irradiated mice were reconstituted by intravenous injection of WT or Rac2–/– fetal liver cells (1 to 2 x 106/mouse) immediately after irradiation. In separate studies, a 50:50 mixture of WT (bearing the CD45.1 allele) and Rac2–/– fetal liver cells (bearing the CD45.2 allele) was given to 12 Gy irradiated WT recipients (1 to 2 x 106 per recipient). Antibiotics were continued for at least 4 weeks, and the mice were kept in microisolator cages in a barrier facility.

Rac2 mRNA Expression

Human mesenchymal stem cells (MSCs, also called human marrow stromal cells) were isolated and cultured as described.13 Total RNA was extracted from MSCs using a QiaShredder and a RNeasy mini kit (Qiagen Inc., Valencia, CA). RNA was reverse-transcribed, and quantitative real-time PCR was performed as described for IL-17. The primer sequences were Rac2: forward 5'-GCAAGACCTGCCTTCTCATCA-3'; reverse 5'-GCTGTCCACCATCACATTGG-3'. A standard curve was constructed using the purified PCR product corresponding to the target sequence of WT Rac2 (1 x 10–5 to 1 x 10–9 ng/ml). The Prism7000 software package generated a standard calibration curve of CT versus known quantities of Rac2 reference DNA, and the starting quantity of Rac2 cDNA per µl of the reverse transcriptase (RT) reaction was calculated.

To measure Rac2 expression in mouse BM cells, total RNA was extracted from BM cells using RNA-Bee (Tel-Test, Friendswood, TX). Rac2 mRNA was quantitated as described for human Rac2, with primers specific for mouse Rac2 (forward: 5'-ACTGTCATCCCTGGCTGCT-3'; reverse: 5'-CAGGTTCACCGGCTTACTG-3'). The concentration was determined based on a standard curve constructed from purified PCR product of the target mouse Rac2 sequence.

In a separate study, BM cells from chimeric mice were magnetically sorted using CD45-coated MACS beads and cell separation columns (Miltenyi Biotec Inc., Auburn, CA). Total RNA was isolated from sorted cells using QiaShredder and RNEasy kit (Qiagen). Rac2 mRNA was quantified by quantitative real-time RT-PCR using ABI Prism 7000 (Applied Biosystems) as described above.

Surface Markers to Determine Leukocyte Subtype and Genotype

The contribution of WT and Rac2–/– stem cells to leukocyte subpopulations was evaluated by staining blood and BM samples with antibodies to CD45.1 or CD45.2, and antibodies to surface markers expressed by neutrophils (Gr-1), B lymphocytes (B220), and T lymphocytes (CD3). Samples were incubated with various combinations of fluorescein isothiocyanate-conjugated anti-CD45.2, phycoerythrin-conjugated antibodies to CD3, B220, or Gr-1, or biotin-conjugated antibodies to CD45.1 or c-kit (CD117), followed by incubation with streptavidin-conjugated phycoerythrin-Cy5 (all reagents from BD Pharmingen). Samples were read using a BD FACScan, and data were processed using CellQuest (BD Pharmingen) and FCS Express (DeNovo Software, Thornhill, Canada).

Statistics

Data were compared using analysis of variance, Mann Whitney U-test, or two-tailed Student’s t-test (paired or independent), as appropriate. Differences between groups in multigroup comparisons were assessed using Scheffé’s post hoc test. Correlation between sets of data were determined using Pearson’s product moment statistic. Differences between groups were considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Neutrophil Kinetics

Rac2–/– animals have persistently elevated numbers of neutrophils in the BM and circulation compared to WT animals. To help clarify the mechanisms that underlie this increase, neutrophil kinetics was examined in Rac2–/– and WT mice using a single injection of BrdU to label dividing cells in the BM. The appearance of BrdU-labeled neutrophils was tracked in the BM and the circulation by measuring the percentage of Gr-1+ cells that incorporated BrdU. In the cohort of mice that received BrdU, circulating neutrophil counts and the proportion of Gr-1+ cells in the BM were significantly elevated in Rac2–/– animals compared to WT animals (Table 1) .


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  		Table 1. Circulating Counts in the Blood and the Gr-1+ Fractions in the BM

 
In the BM, the percentage of Gr-1+ cells that incorporated BrdU is shown in Figure 1 . This proportion was lower in Rac2–/– mice compared to WT animals at 24 and 48 hours after BrdU administration, and declined steadily after 72 hours in both genotypes. The rise in the proportion of Gr-1+ cells that incorporated BrdU in the BM up to 48 hours after BrdU administration likely reflects the up-regulation of Gr-1 expression by maturing myeloid precursors that had taken up BrdU. These results suggest that the increase in the number of Gr-1+ cells in the BM of Rac2–/– mice is not attributable to an increase in the fraction of late-stage myeloid precursors that are dividing.


Figure 1
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  		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).

 
In the circulation, there were few BrdU-labeled neutrophils in the WT and Rac2–/– mice up to 48 hours after BrdU administration (Figure 2A) . By 60 hours, this number had increased in both genotypes and was significantly greater in the Rac2–/– mice through 168 hours. When the percentages of total neutrophils that were BrdU+ were compared, the percentage of BrdU+ circulating neutrophils was lower at early time points (12, 24, and 48 hours after BrdU injection) in Rac2–/– animals compared to WT (Figure 2B) . The percentage of BrdU+ neutrophils was higher in the Rac2–/– than in WT mice by 72 hours and through 144 hours after BrdU administration.


Figure 2
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  		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).

 
BrdU+ circulating neutrophils at early time points incorporated the most BrdU, based on the intensity of their staining with the fluorescein isothiocyanate-conjugated anti-BrdU antibody. BrdU bright cells are likely derived from neutrophil precursors that incorporated BrdU during the S phase of their last division. Consistent with this idea, the BrdU+ circulating neutrophils at early time points were almost entirely BrdUbright. The absolute number and the percentage of BrdUbright circulating neutrophils are shown in Figure 2, C and D , respectively, and indicated similar differences between genotypes as seen for all BrdU+ neutrophils.

The circulating half life of labeled neutrophils was calculated by plotting the time of peak BrdU labeling and thereafter versus the number of circulating BrdUbright neutrophils. An exponential curve was fitted to these data (R2 ≥0.95 in both groups), and the decay constant k was obtained from the equation y = aekx. The half life t1/2 was calculated as t1/2 = ln2/k. The calculated half life was 11.5 hours and 10.4 hours for WT and Rac2–/– BrdUbright neutrophils, respectively. These values are similar to those reported for circulating neutrophils in humans and mice using similar methods.14,15 These calculations indicate that the neutrophilia observed in Rac2–/– mice is not because of an increased neutrophil half-life in the circulation.

Thus, these studies indicate that Rac2 may be required for the timely release of neutrophils into the circulation, because the proportion of BrdU-labeled neutrophils up to 48 hours after BrdU administration was significantly lower in the Rac2–/– than WT mice. The increased number of BrdUbright neutrophils in the Rac2–/– mice compared with WT mice at the later time points after BrdU administration may reflect the delayed release of labeled neutrophils from the enlarged pool of Gr-1+ cells in the BM because there was no significant difference in the circulating half life of WT and Rac2–/– neutrophils.

Circulating G-CSF and Lung IL-17 mRNA Are Elevated in Rac2–/– Mice

Because G-CSF is a major regulator of granulopoiesis and may also mediate neutrophil trafficking from the BM,16,17 the levels of circulating G-CSF were determined in WT and Rac2–/– animals. CD18-deficient mice are reported to have elevated circulating G-CSF levels that correlated with their high circulating neutrophil counts.18,19 The mean circulating neutrophil count in the Rac2–/– mice was approximately six times higher than that in the WT mice (Figure 3A) . Circulating G-CSF levels in Rac2–/– mice were significantly higher than in WT controls (Figure 3B) .


Figure 3
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  		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).

 
IL-17 can induce G-CSF production by fibroblasts and endothelial cells.20 Mice deficient in CD18 or a number of other leukocyte adhesion molecules have elevated neutrophil counts that correlate with increased levels of IL-17 and G-CSF in the circulation,18 and have elevated levels of IL-17 expression in various tissues, including the lung.19 The amount of IL-17 mRNA in the lungs of Rac2–/– mice was greater than in WT mice (Table 2) . The difference in the mean CT between WT and Rac2–/– samples was 5.5 cycles, indicating that the expression of IL-17 mRNA in the lung of Rac2–/– mice was ~ 25.5 or 45 times higher than that seen in the lung of WT mice. These data suggest that increased G-CSF and IL-17 contribute to the neutrophilia observed in Rac2–/– mice.


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  		Table 2. IL-17 mRNA Expression in the Lungs of Rac2–/– Mice Is Significantly Greater than WT Mice

 
Rac2 in Hematopoietic and Nonhematopoietic Cells Can Regulate Neutrophil Production and Release

To determine whether the absence of Rac2 in hematopoietic cells is sufficient for the neutrophilia in Rac2–/– animals, the hematopoietic systems of lethally irradiated WT or Rac2–/– animals were reconstituted with WT or Rac2–/– fetal liver cells. The percentage of BM cells that expressed Gr-1 was higher in the animals that received Rac2–/– stem cells than in animals that received WT stem cells (Table 3) , indicating that missing Rac2 in the hematopoietic stem cells was sufficient to cause the increased production of Gr-1+ cells in the BM.


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  		Table 3. The Percentage of Gr-1+ Cells in the BM and Cell Counts in the BM and Blood of WT and Rac2–/– Recipients 24 Weeks after Receiving WT or Rac2–/– Stem Cells

 
The circulating neutrophil counts of Rac2–/– hosts given Rac2–/– stem cells were significantly higher compared to WT hosts given WT stem cells (Table 3) . The neutrophil counts in WT hosts given Rac2–/– stem cells and in Rac2–/– mice given WT stem cells were intermediate between the two other groups, and not significantly higher than WT mice given WT stem cells. The circulating lymphocyte counts showed similar results (Table 3) , a significant increase seen only when Rac2 was absent in both the donor and the recipient. These data suggest that Rac2 deficiency is required in both the host and the recipient before a significant increase in circulating neutrophils or lymphocytes is observed.

The finding that Rac2 deficiency in the lethally irradiated recipients influenced circulating leukocyte counts was surprising, because Rac2 expression has been described only in cells of hematopoietic origin.1,21,22 To begin to determine whether Rac2 is expressed by BM cells that are not hematopoietic stem cells or their derivatives, Rac2 mRNA was measured by quantitative RT-PCR in cells flushed from the BM of chimeric mice. As expected, abundant Rac2 mRNA was expressed in the BM of WT recipients of WT stem cells and in the BM of Rac2–/– recipients of WT stem cells, and no Rac2 message was detected in Rac2–/– recipients of Rac2–/– stem cells (Figure 4) . Intriguingly, low levels of Rac2 mRNA were detectable in all of the samples from WT recipients of Rac2–/– stem cells, indicating the presence of radioresistant host-derived cells in the BM of these mice. Similar observations were found in WT mice given either 7 or 12 Gy irradiation and Rac2–/– stem cells. These host-derived BM cells may be residual WT hematopoietic cells that survived after irradiation and/or radioresistant nonhematopoietic cells that express low levels of Rac2.


Figure 4
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  		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).

 
To determine whether nonhematopoietic BM stromal cells express Rac2, BM cells were collected from lethally irradiated WT mice reconstituted with Rac2–/– stem cells. The BM cells were magnetically sorted according to CD45 surface expression, and Rac2 message was determined in the CD45+ and CD45– fractions. Interestingly, Rac2 message was detected in the highly purified (>99% CD45-negative) nonhematopoietic fraction of BM cells (Table 4) , indicating that Rac2 is likely expressed by stromal cells in the BM. Low levels of Rac2 message was detected in the CD45+ fraction, indicating that radioresistant recipient-derived hematopoietic cells were present (Table 4) . To confirm that nonhematopoietic cells in the BM express Rac2, Rac2 expression was determined in cultures of BM stromal cells. Because murine BM stromal cells are difficult to isolate and grow in pure culture, human MSCs were studied. Human MSCs are nonhematopoietic marrow stromal cells that can give rise to cells of mesenchymal lineage and support hematopoiesis. Rac2 mRNA was expressed in cultured human MSCs isolated from three donors (Figure 5) , suggesting that human BM stromal cells express this GTPase.


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  		Table 4. Rac2 mRNA Levels in BM Cells from WT Mice Reconstituted with Rac2–/– Fetal Liver Cells

 

Figure 5
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  		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.

 
The Presence of WT Stem Cells Suppresses Neutrophilia in Recipients of WT and Rac2–/– Stem Cells

Because Rac2 is required for many functions of leukocytes, the neutrophilia in the Rac2–/– mice may be attributable to defects in host defense that cause chronic BM stimulation. These extrinsic effects on neutrophil production can be suppressed by the presence of WT leukocytes, presumably by conferring protection to the host. If Rac2 can regulate leukocyte production in a cell-intrinsic manner, however, defects will persist even in the presence of WT leukocytes. To examine whether Rac2 in hematopoietic cells regulates leukocyte production in a cell-intrinsic manner, lethally irradiated WT recipients were given a 50:50 mixture of WT and Rac2–/– fetal liver cells. The presence of WT stem cells gives rise to WT leukocytes that are able to protect the host, and thus obviate the confounding effects of host defense defects on neutrophil production. This approach also allows the comparison of the WT and Rac2–/– neutrophils in the same WT environment. WT mice expressed the CD45.1 allele, whereas Rac2–/– mice expressed CD45.2, allowing the genotype of leukocytes to be distinguished by flow cytometry.

To verify that any observed defects in these chimeric mice are not attributable to differences in the number of stem cells infused, the percentage of fetal liver cells that were lineage-negative, Sca-1+, c-kit+ (LSK, the fraction enriched for hematopoietic stem cells) was determined. The data showed that this percentage was similar in the WT and Rac2–/– donor preparations used to reconstitute the recipients (0.21% versus 0.20%, respectively). To rule out defects attributable to differences in the number of committed hematopoietic progenitors in the donor fetal liver, the chimeric mice were studied at least 20 weeks after transplantation. This interval was selected based on literature demonstrating that at this time donor-derived hematopoietic cells arose from long-term hematopoietic stem cells,23 rather than more differentiated progenitors in the donor fetal liver.23

The circulating neutrophil count in animals that received a 50:50 mixture of WT and Rac2–/– fetal liver cells was similar to that in WT hosts given WT stem cells (0.98 ± 0.2 x 106/ml and 1.0 ± 0.16 x 106/ml, respectively; Table 5 .). The proportion of BM cells that expressed Gr-1 was lower in the mixed chimeric mice than in WT mice given WT stem cells and Rac2–/– mice given Rac2–/– stem cells (Table 5) . These data suggest that the neutrophilia observed in the Rac2–/– animals was primarily attributable to extrinsic effects acting on the BM, because the presence of WT stem cells prevented the increase in neutrophils in the blood and BM.


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  		Table 5. Circulating Counts in the Blood and the Gr-1+ Fractions in the BM

 
Lineage Distribution of Rac2–/– Leukocytes Is Skewed in Recipients of Mixed WT and Rac2–/– Stem Cells

In WT hosts given a 50:50 mixture of WT and Rac2–/– stem cells, Rac2–/– cells comprised only 31 ± 2.6% and 40 ± 4.9% of CD45+ cells in the blood and BM, respectively, rather than the expected 50%. Rac2–/– leukocytes comprised less than half of circulating B and T lymphocytes and less than a third of B cells in the BM in these mice (Table 6) . Intriguingly, the percentage of Gr-1+ cells that were Rac2–/– was 49 ± 6% and 42 ± 4.7% in the BM and blood, respectively, only slightly lower than the expected proportion of 50% (Table 6) . Rac2–/– cells made up 58 ± 5.4% of the c-kit high-expressers in the BM, a population enriched for myeloid precursors. Thus, Rac2 deficiency appears to have a larger detrimental effect on the ability of stem cells to reconstitute lymphoid cells compared with myeloid cells when both WT and Rac2–/– stem cells are present.


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  		Table 6. The Percentage of Leukocytes that Are Rac2–/– in the Blood and BM of Recipients of a 50:50 Mixture of WT and Rac2–/– Fetal Liver Cells

 
The difference in the ability of Rac2–/– stem cells to contribute to the lymphoid and myeloid lineages in mice transplanted with a mixture of WT and mutant stem cells suggests that Rac2 modulates the lineage distribution of leukocyte progenitors/precursors. In the circulation and BM, the fraction of Rac2–/– hematopoietic cells that were Gr-1+ was significantly greater compared to the fraction of WT hematopoietic cells that were Gr-1+ (Figure 6) . Conversely, a lower percentage of circulating Rac2–/– leukocytes was Gr-1-negative (nongranulocytes) compared with WT leukocytes (77 ± 4% versus 86 ± 2.8%, P < 0.05, paired t-test). Thus, Rac2 deficiency appears to alter the lineage distribution of leukocytes in the blood and BM.


Figure 6
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  		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).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Neutrophil numbers in the blood and BM of mice reconstituted with a mixture of WT and Rac2–/– stem cells were lower than those in recipients of Rac2–/– stem cells alone, indicating that neutrophilia in the Rac2–/– mice is attributable to extrinsic factors that can be primarily corrected by the presence of WT leukocytes. We suggest that these extrinsic factors are caused by defects in host defense that result in chronic stimulation of the BM. However, subtle defects in leukocyte lineage distribution and circulating leukocyte counts persist in the presence of WT leukocytes, suggesting that Rac2 can modulate leukocyte lineage distribution. Rac2 deficiency in hematopoietic cells causes an increase in the fraction of neutrophils relative to other leukocytes. In addition, Rac2 appears to be expressed in nonhematopoietic stromal cells, and Rac2 in these cells appears to regulate the production and retention of hematopoietic cells within the BM.

Elevated IL-17 and G-CSF May Contribute to the Neutrophilia Observed in the Absence of Rac2

Rac2–/– mice have elevated levels of G-CSF in their circulation and IL-17 in their lung compared to WT mice. High levels of G-CSF and IL-17 have been shown to correlate with elevated neutrophil counts in mice deficient in CD18 or E/P-selectins.18,19 Circulating neutrophil counts were 6- and 25-fold higher in the Rac2–/– and CD18-deficient mice, respectively, compared to WT mice (Figure 3A and data not shown). Circulating G-CSF levels in Rac2–/– mice are approximately three times those in WT mice (Figure 3B) and approximately half those observed in CD18-deficient mice (824 ± 223 pg/ml), and IL-17 mRNA levels in the lung of Rac2–/– and CD18-deficient mice are similarly elevated compared to IL-17 expression in the lung of WT mice (45-fold and 43-fold, respectively). Spontaneous infections are less common in Rac2–/– mice than in mice lacking CD18 or E/P-selectins, and neutrophilia in Rac2–/– mice is much less severe than in CD18-deficient or E-/P-selectin-null mice,6,10,24 suggesting that host defense defects contribute to the neutrophilia. These observations suggest that the relationship between the levels of IL-17 and G-CSF and the circulating neutrophil counts is complex, and that the number of circulating neutrophils is the result of many integrated events.

Whether Rac2 may also directly modulate IL-17 and G-CSF production or signaling is not known. Rac2 has been shown to play a role in the generation and function of some T lymphocyte lineages,25,26 so Rac2 might likewise regulate IL-17-producing T cells. Rac2 in nonhematopoietic cells may also modulate signaling downstream of the IL-17 receptor. A recent study reported that Rac2 is expressed in vascular smooth muscle cells, where it regulates migration and superoxide production.27 Rac2 is present in endothelial cells (unpublished observations), where it could act downstream of the IL-17 receptor to control G-CSF production.

Rac2 Is Expressed in Nonhematopoietic Cells and Might Modulate Neutrophil Production and Release from the BM

The data in Table 3 and Figures 4 and 5 suggest that nonhematopoietic cells in the BM express Rac2 message, albeit at low levels. Immunohistochemical studies showed low-level expression of Rac2 protein in human MSCs using confocal microscopy (data not shown). However, demonstrating Rac2 protein expression in nonhematopoietic cells using antibody-based techniques is limited by the high homology between Rac1 and Rac2, so that antibodies that recognize Rac2 cross-react to some degree with Rac1, which is expressed at much higher levels than Rac2 in nonhematopoietic cells. Studies are underway to study Rac2 protein expression and function in nonhematopoietic cells using techniques with improved specificity and sensitivity. Recently, Tian and Autieri27 reported Rac2 message and protein expression in vascular smooth muscle cells, providing another instance in which a nonhematopoietic cell expresses Rac2.

The data indicating that Rac2 message is expressed in the BM of WT hosts given Rac2–/– stem cells and that circulating neutrophil numbers are significantly elevated in Rac2–/– hosts given Rac2–/– stem cells but not in other groups suggest that Rac2 expressed in recipient-derived cells can regulate the number of circulating leukocytes. The identity of these recipient-derived regulatory cells is not known. Stromal cells in the BM are good candidates as regulatory cells because they directly contact hematopoietic cells and regulate hematopoietic cell growth and development, and cultured human MSCs express Rac2 message. Cultured human MSCs can differentiate into cells belonging to various mesenchymal lineages and can support in vitro hematopoiesis.28 Rac2 in stromal cells may be required to anchor hematopoietic cells within the BM, or it may be required for stromal cell-derived signals that support hematopoiesis. Alternatively, Rac2 may be expressed in other nonhematopoietic cells, perhaps in the BM endothelial cells that line the venous sinusoids. Our studies do not completely rule out the presence of a small number of residual radioresistant recipient-derived hematopoietic cells that may be able to regulate neutrophil counts. The highest dose of lethal irradiation given in these studies was 12 Gy, which is lethal when no BM reconstitution is given. Even at this radiation dose, however, a small number of recipient-derived hematopoietic cells persist in the BM or in other organs for some time. Whether these rare residual hematopoietic cells can regulate neutrophil counts is not known. It is possible that the persistence of a rare population of WT cells in the WT hosts could limit neutrophilia in the recipients of Rac2–/– stem cells, whereas residual Rac2–/– cells in the Rac2–/– hosts of WT stem cells could account for slightly elevated neutrophil numbers in these mice. This scenario seems unlikely, however, because in recipients of a 50:50 mixture of WT and Rac2–/– stem cells, the proportion of WT to Rac2–/– neutrophils is close to 50:50 in the circulation, and the total neutrophil counts are similar to WT mice reconstituted with WT stem cells.

The studies examining neutrophil trafficking in WT and Rac2–/– mice suggest that Rac2 may play a complex role in the timely release of neutrophils into the circulation, because the proportion of BrdU-labeled neutrophils up to 48 hours after BrdU administration was significantly lower in the Rac2–/– than WT mice, and a steep rise in the number and proportion of BrdU+ neutrophils occurs in the circulation of Rac2–/– mice beginning between 48 and 60 hours after BrdU administration, ~12 hours earlier than in WT. It is unclear whether these effects are mediated by Rac2 expressed in hematopoietic cells or nonhematopoietic cells or both. Rac2 in hematopoietic cells may regulate the ability of mature neutrophils to migrate from the stroma and enter into the venous sinusoids. In nonhematopoietic cells, Rac2 may regulate the fenestrae in the sinusoidal wall through which leukocytes pass to enter the circulation and/or regulate the interactions between leukocytes and stromal cells. Rac2 may also directly regulate the level of cytokines such as G-CSF that control neutrophil trafficking in many cell types.

Alteration of Lineage Distribution in Rac2–/– Leukocytes

Rac2 in hematopoietic cells has been reported to regulate the growth and differentiation of T and B lymphocytes and other hematopoietic cells.29 Jansen and colleagues30 showed that Rac2–/– HSC/P adhered less to BM stromal cells in vitro and exhibited growth defects in stroma-dependent cultures, indicating that Rac2 in hematopoietic cells is required for their optimal growth and development. Although Rac2 deficiency leads to an increase in circulating HSC/P numbers, Rac2 is not required in the homing and engraftment of transplanted HSC/Ps in the BM after lethal irradiation.31 Rac2–/– hematopoietic cells show growth and survival defects in vitro. Apoptosis is increased in Rac2–/– HSC/Ps compared to WT after stimulation with SCF in vitro.9 Myeloid colony growth in response to GM-CSF is defective in Rac2–/– BM cells.9,32 Surprisingly, this defect is rescued by transducing Rac2–/– cells with constructs encoding mutant forms of Rac2, suggesting that proliferation and survival signals mediated by Rac2 are independent of its functions in motility or superoxide generation.32 However, the neutrophilia observed in Rac2–/– mice indicate that Rac2-independent processes of neutrophil production can compensate for the growth and survival defects described in Rac2–/– progenitors.

When lethally irradiated recipients were given a 50:50 mixture of WT and Rac2–/– fetal liver stem cells to reconstitute their hematopoietic systems, Rac2–/– cells comprised nearly half of Gr-1+ cells in the blood and BM (Tables 5 and 6) , but a much lower percentage of circulating lymphocytes (Table 6) . A greater percentage of Rac2–/– circulating leukocytes were neutrophils compared with WT leukocytes that were neutrophils (Figure 6) . These observations are unlikely to be attributable to defects in BM homing or short-term engraftment of Rac2–/– stem cells, which has been shown to be similar to WT stem cells.30,31 Long-term engraftment of Rac2–/– stem cells in WT recipients is defective30,31 ; however, this stem cell defect would affect both lymphoid and myeloid lineages and may not explain the differential effect in myeloid versus lymphoid cells described here. The skew in the lineage distribution of Rac2–/– leukocytes may indicate that the absence of Rac2 in hematopoietic progenitors favors commitment toward the neutrophil lineage, or Rac2 deficiency may result in increased proliferation of committed neutrophil progenitors/precursors. However, the deficiency in Rac2 does not appear to increase the proportion of late-stage neutrophil precursors that are proliferating (unpublished observations), and Rac2–/– LSK cultured under conditions that support myeloid development do not give rise to more neutrophils compared to WT LSK (unpublished observations). These observations suggest that the increased proportion of neutrophils in Rac2–/– leukocytes likely reflects a requirement for Rac2 in the development and maintenance of lymphocytes or their precursors. Indeed, Rac2 is required for B-cell development and maintenance,33,34 primarily by mediating receptor-driven survival signals. In T cells, Rac2 has been shown to regulate differentiation and activation of T-helper cell subsets.26,35 Thus, the absence of Rac2 in hematopoietic cells appears to differentially affect lymphoid and myeloid cells.

Rac2 deficiency results in a modest several-fold increase in circulating neutrophil counts. In comparison, CD18-deficient mice have circulating counts that are many times higher than those seen in WT mice.10 This difference in the degree of neutrophilia may be attributable to the expression of Rac1, which may partly compensate for the loss of Rac2 in Rac2–/– mice. However, Rac2-specific functions in neutrophils have been reported, particularly in the ability to respond to fMLP and to activate NADPH oxidase in response to inflammatory stimuli.6,9,36,37 The requirement for Rac2 in the survival of HSC/Ps and more differentiated myeloid precursors suggests that intrinsic mechanisms may partially limit the extent of neutrophilia in Rac2–/– animals caused by defects in host defense and systemic or paracrine effects on the BM.

The data presented here suggest that Rac2 has multiple roles in regulating neutrophil production and release from the BM. Rac2 in hematopoietic cells regulates neutrophil production indirectly, through its role in mediating neutrophil recruitment and activation for host defense. Rac2 in hematopoietic cells directly regulates the lineage distribution of leukocytes, primarily affecting lymphocyte production. Rac2 expressed in nonhematopoietic cells may participate in modulating circulating neutrophil counts.


   Acknowledgements
 
We thank Omer Koc, M.D., Nancy A. Rebert, Sarah Richer Dawson, and Emese Szekely for expertise and helpful discussions.


   Footnotes
 
Address reprint requests to Claire M. Doerschuk, M.D., Department of Medicine, University of North Carolina at Chapel Hill, CB #7248, 7011 Thurston-Bowles, Chapel Hill, NC 27599-7248. E-mail: cmd{at}med.unc.edu

Supported by the Public Health Service (grants R37HL048160, RO1HL077370, and T32HL007415).

Accepted for publication April 29, 2008.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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