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The Journal of Immunology, 2006, 177: 8512-8521.
Copyright © 2006 by The American Association of Immunologists, Inc.

Defective Chemokine-Directed Lymphocyte Migration and Development in the Absence of Rho Guanosine Diphosphate-Dissociation Inhibitors {alpha} and beta1

Hiroyoshi Ishizaki*,{dagger}, Atsushi Togawa{ddagger}, Miki Tanaka-Okamoto*, Keiko Hori*, Miyuki Nishimura{dagger}, Akiko Hamaguchi{dagger}, Toshio Imai{dagger}, Yoshimi Takai§ and Jun Miyoshi2,*

* Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan; {dagger} KAN Research Institute, Kyoto, Japan; {ddagger} Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan; and § Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Rho family small GTP-binding proteins, including Rho, Rac, and Cdc42, are key determinants of cell movement and actin-dependent cytoskeletal morphogenesis. Rho GDP-dissociation inhibitor (GDI) {alpha} and Rho GDIbeta (or D4/Ly-GDI), closely related regulators for Rho proteins, are both expressed in hemopoietic cell lineages. Nevertheless, the functional contributions of Rho GDIs remain poorly understood in vivo. In this study, we report that combined disruption of both the Rho GDI{alpha} and Rho GDIbeta genes in mice resulted in reduction of marginal zone B cells in the spleen, retention of mature T cells in the thymic medulla, and a marked increase in eosinophil numbers. Furthermore, these mice showed lower CD3 expression and impaired CD3-mediated proliferation of T cells. While B cells showed slightly enhanced chemotactic migration in response to CXCL12, peripheral T cells showed markedly reduced chemotactic migration in response to CCL21 and CCL19 associated with decreased receptor levels of CCR7. Overall, Rho protein levels were reduced in the bone marrow, spleen, and thymus but sustained activation of the residual part of RhoA, Rac1, and Cdc42 was detected mainly in the bone marrow and spleen. Rho GDI{alpha} and Rho GDIbeta thus play synergistic roles in lymphocyte migration and development by modulating activation cycle of the Rho proteins in a lymphoid organ-specific manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Small GTP-binding proteins of the Rho family, Rho, Rac, and Cdc42 are key signal transducers that regulate cell adhesion and migration, as well as multiple cellular activities, including proliferation, apoptosis, and transcription (1, 2, 3). The Rho family proteins regulate the reorganization of the actin cytoskeleton and the integrity of associated integrin adhesion complexes. Rac facilitates the formation of lamellipodia protrusions and membrane ruffles at the leading edge of migrating cells, Cdc42 triggers filopodial extensions at the cell periphery, and Rho regulates stress fiber and focal adhesion assembly (1, 4). As with hemopoietic cells, Rac supports cell cycling, migration, short-term homing, while Cdc42 is necessary for maintaining cellular polarity and yet is dispensable for sensing and crawling toward inflammation sites. Rho coordinates down-regulation of cell migration and is required for cells to retract from sites of matrix- and cell-cell contacts (5, 6). All these cytoskeleton-dependent activities are essential for lymphocyte activation and the interaction between lymphoid cell-cell contact sites. Therefore, Rho family proteins are recognized as pivotal regulators of lymphocyte motility and polarization, Ag-specific T cell activation, and immunological synapse formation (7).

Chemokines orchestrating lymphocyte migration are essential for activation of immune responses (8, 9). For example, the chemokine CXCL12 (SDF-1{alpha}) induces Rac activation that mediated microvillar breakdown, leading to T lymphocyte emigration and a fruitful NK cell activity and polarization state (10). CXCL12 also controls efficient up-regulation of the integrin 41-dependent T lymphocyte adhesion (11). Inhibition of Rac-mediated membrane ruffling and Rho-mediated Rho kinase activation results in the inhibition of chemotaxis of hemopoietic cells (12). Although studies on Rac have demonstrated the roles in lymphocyte development in mouse models (5, 13, 14, 15, 16, 17), it remains unclear how cooperatively Rho family proteins are involved in cell movement accompanied by directional sensing such as chemokine-directed lymphocyte migration in vivo, and how they have active contributions to establishing the pattern of cell migration in lymphoid organs.

Rho GDP-dissociation inhibitors (GDI)3 bind to various Rho proteins and regulate their function in three ways. First, Rho GDIs block activation of Rho proteins by sequestering the GDP-bound Rho proteins in the cytosol, thereby inhibiting the exchange of GDP to GTP of Rho proteins (18). Second, Rho GDIs target Rho proteins to specific signaling complexes at the plasma membrane and facilitate coupling Rho proteins with their downstream effector proteins (19). Finally, Rho GDIs protect Rho proteins from proteolytic degradation by forming stable complexes in the cytosol. These roles of Rho GDIs are important to understand consequences of Rho GDI disruption that may result initially in activation of the Rho family, but later in a blockade of activation because of increased Rho protein degradation.

Rho GDIs comprise three isoforms: Rho GDI{alpha} (Rho GDI-1), Rho GDIbeta (or D4/Ly-GDI), and Rho GDI{gamma} (Rho GDI-3). Rho GDI{alpha} and Rho GDIbeta are cytosolic proteins similar in structure and function (20, 21, 22, 23), whereas the noncytosolic Rho GDI{gamma} seems to have a function slightly different from those of Rho GDI{alpha} and Rho GDIbeta (24). Rho GDI{alpha} is ubiquitously expressed in mouse tissues, while Rho GDIbeta is exclusively expressed in hemopoietic cells. We have previously shown Rho GDI{alpha}-deficient mice developed degeneration of renal tubular epithelial cells and impaired spermatogenesis, but they showed no apparent phenotype affecting lymphocyte functions (25). Similarly, Rho GDIbeta-deficient mice showed the minimal alterations of the immune responses (26). Therefore, combined deficiencies of Rho GDI{alpha} and Rho GDIbeta could provide us with experimental conditions in which membrane-cytosol shuttling processes of all Rho proteins are selectively impaired in lymphoid cell lineages. In this study, we report that Rho GDI{alpha} and beta double-null mutant mice exhibit aberrant homeostasis of lymphocyte development such as displacement of splenic marginal zone (MZ) B cells, retention of T cells in the thymic medulla, and increased eosinophil population. Our study suggests that Rho GDI{alpha} and Rho GDIbeta play synergistic roles in lymphocyte migration and development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of Rho GDI{alpha}beta–/– mice

Rho GDI{alpha}–/– mice were null mutants as previously described (25). Rho GDIbeta–/– mice were newly generated by replacing the 1.7-kb DNA region covering the promoter region and the exons 1–2 of the Rho GDIbeta gene with an MC1-promoted neomycin-resistant gene cassette by homologous recombination in CCE embryonic stem cells. CCE cells with the targeted mutation of the Rho GDIbeta gene were identified by Southern hybridization using the 5' and 3' probes as indicated (see Fig. 1A), and were injected into C57BL/6 blastocysts. Chimeric mice were generated as described (25). Male chimeric mice were mated with female BDF1 (F1 hybrid of C57BL/6 and DBA/2) mice. Genotyping of mice with agouti coat color were performed by Southern hybridization using tail DNA digested by XbaI and EcoRI and/or PCR analyses. PCR primers (Rho GDI{alpha}, 5'-AGAACAGGAACCCACTGCTGAGCA-3' and 5'-AGTGCTGCATACCAAGGTCAGTCG-3'; Rho GDIbeta, 5'-AGCTCAATTATAAGCCACCCC-3' and 5'-GACATCTCCCAGCAGTGTTTT-3'), located in the DNA region replaced by the neomycin-resistance gene, are used to amplify a 380-bp band for wild-type (WT) Rho GDI{alpha} allele (25) and a 107-bp band for the WT Rho GDIbeta allele. Primers 5'-GGGCGCCCGGTTCTTTTTGTC-3' and 5'-GCCATGATGGATACTTTCTCG-3' were used to amplify a 224-bp band derived from the neo-resistance gene to detect the mutant allele. Animal experiments were conducted in accordance with protocols approved by the Osaka University Committee for the Care and Use of Laboratory Mice. This study has been approved by the Review Committee of the Osaka Medical Center for Cancer and Cardiovascular Diseases.


Figure 1
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  		FIGURE 1. Impaired spermatogenesis of Rho GDI-deficient mice. A, Targeting strategy generating Rho GDIbeta–/– mice is schematically shown. B, Genotyping by PCR analysis of tail DNA extracted from littermate mice at 21 days of age. C, Detection of Rho GDI{alpha} and Rho GDIbeta proteins by immunoblot analyses in splenic B cells (left panel) and T cells (right panel) of each genotype. D, Comparison of spermatogenesis among the testes of 16-wk-old mice lacking Rho GDI{alpha}, Rho GDIbeta, and Rho GDI{alpha}beta. Testes sections are stained with H&E. Scale bars, 100 µm. The degenerative changes are becoming exacerbated corresponding to Rho GDI deficiencies in this order: WT, Rho GDIbeta, Rho GDI{alpha}, and Rho GDI{alpha}beta.

 
Immunoblot analysis

Cell extracts were prepared as described (25). The following Abs were used for immunoblot analysis: anti-RhoA, anti-Cdc42, and anti-{alpha}-tubulin Abs were obtained from Santa Cruz Biotechnology, anti-Rac1 Abs were obtained from BD Transduction Laboratories, and anti-beta-actin Abs were obtained from Sigma-Aldrich.

Flow cytometry

Lymphocyte populations were characterized by using FITC-, PE-, allophycocyanin-, or PerCP-conjugated mAbs and analyzed by FACSCalibur flow cytometer (BD Biosciences) and Flowjo software (TreeStar; Stanford University, Stanford, CA). Abs specific for each marker were purchased as follows: CD45R (B220), CD8, CD11a, CD44, and CD62L were from eBioscience, CD2, CD4, CD19, CD21/CD35, CD23, IgM and IgD were from BD Pharmingen, CD69, Mac-1 (CD11b), and Gr-1 were from Caltag Laboratories, CCR7 was from BioLegend, and CD3 was from Immunotech, and eBioscience. Thymocytes were stained with propidium iodide (Sigma- Aldrich) and annexin V (BD Pharmingen) to detect apoptosis.

Immunohistochemistry

Thymus and spleen samples were snap-frozen in OCT compound (Sakura Finetek) and were stored at –70°C. Cryostat sections from the thymus were fixed with 90% methanol and acetone, blocked using an avidin/biotin blocking kit (Vector Laboratories), and stained with biotin-conjugated Abs for CD4. Biotinylated primary reagents were detected using streptavidin-Texas Red (BD Pharmingen). Anti-CD8 Abs (BD Pharmingen) were detected with anti-rat Alexa Fluor 488 (Molecular Probes). Spleen sections were stained with the following combinations: MOMA-1 (anti metallophilic macrophages) mAb (BMA), anti-Rat IgG Abs coupled to alkaline phosphatase (Zymed Laboratories), the VectorBlue Alkaline Phosphatase Substrate kit (Vector Laboratories), biotin-conjugated anti-CD45R Abs (Cedarlane Laboratories), extravidin peroxidase (Sigma-Aldrich), and the Nova RED Substrate kit for Peroxidase (Vector Laboratories). Bone marrow (BM) and lung sections were stained by using the EoProbe Kit (BioFX Laboratories) for the histological detection of eosinophils.

In vitro lymphocyte proliferation and migration assays

B and T cells were affinity purified using the MACS isolation kit (Miltenyi Biotec), according to the manufacturer’s instruction. Proliferation assays were performed at 5 x 105 cells/ml for B cells and at 2.5 x 105 cells/ml for T cells in 0.2 ml of RPMI 1640 with 10% FCS. B cells were stimulated with 0.1–100 µg/ml LPS (Sigma-Aldrich), and T cells were stimulated with 2 µg/ml anti-CD28 Abs (BD Pharmingen) and with 1–10 µg/ml anti-CD3 Abs (BD Pharmingen) immobilized to 96-well plates. Proliferation on day 2 was measured as BrdU incorporation following a 6-h pulse. Migration assays were performed in Transwell plates of 6.5-mm diameter with 5-µm pore filters (Costar). In brief, 5 x 105 cells in 0.1 ml of the medium were added to the upper chamber, and 0.6 ml of assay medium with none, 100, 300, 500, or 1000 ng/ml of the recombinant mouse chemokines CXCL12, CCL21, CCL19, and CXCL13 (Genzyme) was added to the lower chamber as indicated by the legends for Fig. 5. A sample containing 2.5 x 104 cells diluted in the assay medium was kept as input control for quantitation of the number of migrated cells. The Transwell plates were incubated at 37°C in 5% CO2 for 3 h. The cells migrated to the lower chamber were collected and analyzed with FACSCalibur and Flowjo software.


Figure 5
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  		FIGURE 5. Effect of Rho GDI{alpha}beta deficiency on in vitro proliferation of splenic B and T cells, apoptosis of thymocytes, and migration activities of Rho GDI{alpha}beta–/– lymphocytes toward chemokines. A, The frequency of BrdU-positive B cells from the spleen of WT ({blacksquare}) and Rho GDI{alpha}beta–/– mice ({square}) at 6 wk of age. B cells are cultured for 2 days, treated with LPS at the indicated concentrations, and pulsed with BrdU for 6 h. Incorporation of BrdU was measured as OD by ELISA. *, p < 0.05; **, p < 0.01. B, The frequency of BrdU-positive CD4+ SP T cells from the spleen of WT and Rho GDI{alpha}beta–/– mice at 6 wk of age. T cells are treated with anti-CD3 Abs at the indicated concentrations in the presence of 2 µg/ml anti-CD28 Abs and pulsed with BrdU. *, p < 0.05; **, p < 0.01. C, Apoptosis of thymocytes from WT and Rho GDI{alpha}beta–/– mice. Thymocytes of DN, DP, CD4+ SP, and CD8+ SP populations from 4-wk-old mice (n = 5) are stained with propidium iodide (PI) and annexin V (Ann-V)-FITC, and analyzed by flow cytometry. D, Migration of B cells from the spleen in response to CXCL12 and CXCL13. B220+ cells from WT or Rho GDI{alpha}beta–/– mice at 6 wk of age are used in transwell chemotaxis assays to assess their chemotactic properties toward the medium supplemented with 100, 300, and 500 ng/ml of each chemokine. *, p < 0.05. Bars show the mean SD (n = 4/group). E, Migration of T cells from the spleen in response to the chemokines. CD4+CD8+CD11b– T cells from WT or Rho GDI{alpha}beta–/– mice at 7–9 wk of age are used in Transwell chemotaxis assays. T cells from Rho GDI{alpha}beta–/– mice show a significantly reduced responsiveness to CXCL12 (n = 5), CCL21 (n = 4), and CCL19 (n = 4) compared with those from WT mice. *, p < 0.01; **, p < 0.001. F and G, Migration of CD4+ SP cells (F) and CD8+ SP cells (G) from the thymus in response to CXCL12, CCL21, and CCL19 at indicated concentrations. H and I, Immune-fluorescent profiles of CCR7 receptors in T cells from the spleen (H) and CD4+ SP thymocytes (I) from WT and Rho GDI{alpha}beta–/– mice (n = 6) at 4–7 wk of age.

 
GST pull-down assay of Rho proteins

RhoA, Rac1, or Cdc42 activity was assessed using the RhoA-binding domain of Rhotekin and the Rac1- and Cdc42-binding domain of P65 p21-activated kinase, respectively, according to the manufacturer’s instruction (Upstate Biotechnology). Lymphocytes from the thymus and spleen were washed with cold PBS and suspended in a lysis buffer containing 50 mM Tris-Cl (pH 7.2), 1% Triton X-100, 0.5% sodium deoxycholate, 1% SDS, 500 mM NaCl, 10 mM MgCl2, 1 mM PMSF fluoride, and 10 µg/ml aprotinin and leupeptin, followed by immediate centrifugation at 13,000 x g for 10 min. The extracts were diluted by 10-fold with the SDS-free buffer of the same components and incubated at 4°C for 45 min with glutathione beads coupled with each fusion protein of GST and Rho protein-binding domains and then washed three times. The RhoA, Rac1, or Cdc42 content in these samples was determined by immunoblotting using Abs for these Rho proteins.

Quantitative RT-PCR

To elucidate mRNA expression profiles of RhoA, Rac1, and Cdc42 in the spleen and thymus, RT-PCR was performed with Rho family protein-specific primers derived from mouse cDNA sequences (RhoA, 5'-TGGTTGGGAACAAGAAGGAC-3' and 5'-TGGTCTTTGCTGAACACTCC-3'; Rac1, 5'-AACCTGCCTGCTCATCAGTT-3' and 5'-TTGTCCAGCTGTGTCCCATA-3'; Cdc42, 5'-TTGTTGGTGATGGTGCTGTT-3' and 5'-TGGCTCTCCACCAATCATAA –3'). TaqMan Rodent GAPDH Control Reagents (Applied Biosystems) were used as controls. Total RNA was isolated from thymocytes and splenocytes using TRIzol (Invitrogen Life Technologies) extraction and the RNeasy Mini kit (Qiagen). First-strand cDNA synthesis was performed using a TaKaRa RNA PCR kit (AMV; TaKaRa Bio) according to the manufacturer’s protocol. Real-time PCR amplification was performed using QuantiTect SYBR Green PCR (Qiagen). Data were analyzed using the 7500 Fast Real-Time PCR System (Applied Biosystems).

Statistical analysis

Statistical significance of the difference between two sample groups was assessed by the Student test. Significance was defined as a p value ≤0.05. Values are indicated as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
General properties of Rho GDI{alpha}beta–/– mice

Rho GDI{alpha}beta–/– double-null mice were derived from Rho GDI{alpha}–/– mice (25) and Rho GDIbeta–/– mice that were newly generated (Fig. 1, A and B). Importantly, targeted disruption of lymphoid-specific Rho GDIbeta resulted in neither age-dependent degeneration of kidney function nor male infertility as observed with Rho GDI{alpha}–/– mice (25). The specific disruptions of Rho GDI{alpha} and Rho GDIbeta genes were confirmed by Western blot analyses using extracts from splenic B cells and T cells (Fig. 1C). Disrupting Rho GDI{alpha} or Rho GDIbeta isoform did not alter the protein levels of its counterpart, Rho GDIbeta or Rho GDI{alpha}, respectively, suggesting that there was no compensation between Rho GDI{alpha} and Rho GDIbeta protein levels in the lymphocytes. Rho GDI{alpha}beta–/– mice were exclusively obtained by in vitro or in vivo fertilization and oviduct transfer using Rho GDI{alpha}+/– mice on the Rho GDIbeta–/– background because natural mating of Rho GDI{alpha}+/– Rho GDIbeta–/– mouse pairs yielded no sibling (Table I). Approximately 6.5% of pups were identified as Rho GDI{alpha}beta–/– mice. Although nearly 75% of Rho GDI{alpha}beta–/– mice died during embryogenesis, those that survived in embryonic environments were born and developed normally up to >12 mo in specific pathogen-free conditions. Rho GDI{alpha}beta–/– mice were totally infertile; histologically, Rho GDI{alpha}beta–/– testes showed drastic testicular degeneration (Fig. 1D) compared with Rho GDI{alpha}–/– and Rho GDIbeta–/– testes. Therefore, Rho GDI{alpha} and Rho GDIbeta play synergistic roles in maintaining the male reproductive function.


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  		Table I. Generation of Rho GDI{alpha}beta–/– mice by heterozygous intercrossing for Rho GDI{alpha} on the background of Rho GDIbetanull mutation

 
Aberrant distribution of B cell population in Rho GDI{alpha}beta–/– mice

Since expression of Rho GDI{alpha} and Rho GDIbeta overlaps in the hemopoietic cell lineages, we first analyzed the number and distribution of B cells in Rho GDI{alpha}–/–, Rho GDIbeta–/–, and Rho GDI{alpha}beta–/– mice. The weight and total cellularity of BM, thymus, and lymph nodes were not different among these mice. Those of spleen, however, were reduced exclusively in Rho GDI{alpha}beta–/– mice (p < 0.05, data not shown). Analysis of BM from Rho GDI{alpha}beta–/– mice showed no significant decrease in total leukocyte number. The proportion and numbers of pro-B (IgM–IgD–CD19+CD2–) and pre-B (IgM–IgD–CD19+CD2+) cells did not differ significantly between WT and Rho GDI{alpha}beta–/– BM at 6–7 wk of age, indicating that the early stages of B cell development are mostly unimpaired although some defective B cell maturation was observed in Rho GDI{alpha}beta–/– BM at 12–32 wk of age (Table II).


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  		Table II. Lymphocyte and blood cell populations in WT and Rho GDI-deficient micea

 
B cell numbers of the spleen were reduced by 40% in Rho GDI{alpha}beta–/– mice at 5–7 wk of age; furthermore, they were reduced by 65% in 12- to 26-wk-old mice. To clarify the nature of the reduction, we analyzed B cell subpopulations by flow cytometry. We subdivided splenic B cells from Rho GDI{alpha}beta–/– mice into populations of newly formed (NF; B220+CD23–CD21–), transitional 1 (T1; CD23–CD21lowIgMhighIgDlow), transitional 2 (T2; CD23+CD21highIgMhighIgDhigh), follicular (FO; CD23+CD21intIgMlowIgDhigh), and MZ (CD23–CD21highIgMhighIgDlow) cells. These analyses revealed that subsets of MZ B cells were drastically reduced in Rho GDI{alpha}beta–/– mice compared with those of WT mice at 12 wk of age (Table II and Fig. 2A), whereas NF, T1, T2, and FO cells were moderately reduced by 20–30% in Rho GDI{alpha}beta–/– mice. This near absence of the MZ B cell population was confirmed by immunohistochemistry (Fig. 2B); the localization of B cells was disorganized in the border region of the white pulp of the Rho GDI{alpha}beta–/– spleen. The distribution of MOMA-1-positive metallophilic MZ macrophages appeared to be unimpaired in Rho GDI{alpha}beta–/– mice, suggesting that the absence of MZ B cells is not due to a structural defect that prevents B cells from accumulating in this zone but due to an insufficiency of MZ B cells.


Figure 2
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  		FIGURE 2. Reduction of splenic MZ B cells and accumulation of MLN B cells from Rho GDI{alpha}beta–/– mice. A, Expression of CD21/CD35 and CD23 on B220+ splenic B cells from WT and Rho GDI{alpha}beta–/– mice at 12 wk of age. Gates identify MZ B cells (CD21/CD35+CD23–) and other B lineage cells (CD21/CD35–CD23– and CD21/CD35+CD23+). Dot plots of B200+-gated cells in representative samples are shown (n > 3), and numbers represent percentages of NF, MZ, T1/T2 stage (T1/T2), and FO B cells. B, Immunohistochemistry of spleen sections stained with Abs for MOMA-1 (blue) and B220 (red). The MZ is the part of the parenchyma that lies between the white and red pulps of the spleen. The positions of the FO zone (F), marginal sinus (MS), and MZ are shown in the upper panels. MZ B cells (MZB) and MZ macrophages (MF) are shown in the lower panels. Scale bars, 200 µm. MZ B cells are colocalized with MOMA-1-positive macrophages surrounding B cell follicles of the white pulp in WT mice (left), but are scarce in Rho GDI{alpha}beta–/– mice (right). C, Lymphocytes of MLN from WT and Rho GDI{alpha}beta–/– mice at 23 wk of age are divided into the CD3+ T cells and B220+ B cells. T cell numbers as well as expression levels of CD3 are reduced in Rho GDI{alpha}beta–/– mice compared with WT mice, while B cell proportion is increased in Rho GDI{alpha}beta–/– mice.

 
In contrast, B cell numbers in the mesenteric lymph nodes (MLN) were increased by 2- to 3-fold in Rho GDI{alpha}beta–/– mice (Table II and Fig. 2C). Immunohistochemistry of the MLN showed increases in the size and cellular densities of B220+ cell follicles as well as decreases in those of T cell area (data not shown). Thus, Rho GDI{alpha}beta proteins differentially regulated the B cell populations in secondary lymphoid organs.

Aberrant distribution of T cell population in Rho GDI{alpha}beta–/– mice

We further analyzed the number and distribution of T cells in Rho GDI-deficient mice (Table II and Fig. 3). Relative populations of circulating CD3+ cells in peripheral blood were reduced by 10, 44, and 89% in Rho GDI{alpha}–/–, Rho GDIbeta–/–, and Rho GDI{alpha}beta–/– mice, respectively, when compared with WT controls (Fig. 3A). Thus, the reductions in peripheral T cell population correlated with the severity of Rho GDI deficiencies; furthermore, a marked reduction by 80–90% in splenic T cell population was observed in Rho GDI{alpha}beta–/– mice (Table II). We then analyzed CD3+ cells in the spleen by surface expression of CD11a and CD44. The population of CD11a+CD44low cells was reduced to 14.8% in Rho GDI{alpha}beta–/– mice, indicating loss of naive T cells (Fig. 3B). The numbers of CD3+ cells were also decreased in the MLN of Rho GDI{alpha}beta–/– mice (Fig. 2C). However, the total numbers of thymocytes in Rho GDI{alpha}beta–/– mice were comparable to those of WT mice, and the size and weight of the thymus were not different between WT and Rho GDI{alpha}beta–/– mice. When the thymocytes were sorted for CD4 and CD8, the proportion of CD4+CD8+ DP cells was relatively reduced by 30%, while those of CD4+ single-positive (SP) cells and CD8+ SP cells were elevated 2-fold in Rho GDI{alpha}beta–/– mice (Table II and Fig. 3C). Characteristically, T cell populations in Rho GDI{alpha}beta–/– mice showed lower CD3 levels in peripheral blood (Fig. 3A) and all the lymphoid organs examined, as representatively shown in the MLN (Fig. 2C). The reasons for this are currently unclear. However, this finding as well as the aberrant proportion of CD4+ SP and CD8+ SP cell populations suggest that T cells in Rho GDI{alpha}beta–/– mice have insufficient capabilities for intrathymic development.


Figure 3
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  		FIGURE 3. Defective T cell development and accumulation of CD4+ SP cells in the thymic medulla of Rho GDI{alpha}beta–/– mice. A, Peripheral T cell populations in WT and Rho GDI-deficient mice of each genotype at 18 wk of age (n > 3). Histograms with percentages of T cells expressing CD3 are shown. Data are displayed as dot plots with logarithmic scale. B, CD3+-gated cells from the spleen are sorted for two populations: memory T cells (CD11a+CD44+) and naive T cells (CD11a+CD44low). C, Increases in CD4+ SP and CD8+ SP cells from Rho GDI{alpha}beta–/– mice at 18 wk of age. Thymocyte suspensions from WT and Rho GDI-deficient mice are stained with fluorescent Ab conjugates as indicated. Flow cytometry profiles with percentages of thymocyte populations are shown for representative samples. D, Immunohistochemistry of thymus sections stained with anti-CD4 (red) and anti-CD8 (green) Abs. The images are merged. Cryosections are prepared from WT and Rho GDI{alpha}beta–/– mice at 12 wk of age. The boundaries between the cortex and medulla of the thymus are indicated. Scale bars, 200 µm. E, Increased population of late medullary T cells from Rho GDI{alpha}beta–/– mice at 7 wk of age. Dot plots of CD4+-gated cells in representative samples are shown (n > 3), and numbers represent percentages of CD69+CD62L– early medullary (EM) thymocytes and CD69–CD62L+ late medullary (LM) thymocytes. The total numbers of EM cells are 4.8 x 106 (WT) and 5.6 x 106 (Rho GDI{alpha}beta–/–). Those of LM cells are 2.7 x 106 (WT) and 12.0 x 106 (Rho GDI{alpha}beta–/–). F, Immunohistochemistry of spleen sections stained with anti-CD4 (red) and anti-CD8 (green) Abs. Cryosections are prepared from WT and Rho GDI{alpha}beta–/– mice at 12 wk of age. Scale bars, 200 µm.

 
We next addressed intrathymic distribution of T cells in the Rho GDI{alpha}beta–/– mice. Immune fluorescence showed CD4+CD8+ double-positive (DP) cells were evenly distributed in the inner and outer cortex of the thymus (Fig. 3D), consistent with the restored CD4+CD8+ DP cell population (Fig. 3C). It is of note, however, that mature T cells, mostly comprised of CD4+ SP cells, accumulated in the medulla of the Rho GDI{alpha}beta–/– thymus, whereas WT CD4+ SP cells seemed to constantly emigrate from the medulla toward peripheral blood (Fig. 3D). To determine the developmental stage of the accumulated T cell population, we used anti-CD69 and anti-CD62L Abs to subdivide the T cells into early and late medullary populations. The analysis showed that predominant cells were late medullary cells (CD4+CD69–CD62Lhigh) rather than early medullary cells (CD4+CD69+CD62Llow) (Fig. 3E). In addition, the CD4+CD69–CD62Llow cell population was increased. We presume this population probably reflected a secondary degenerative change in CD62L expression levels of late medullary cells that were unable to emigrate from the thymus. In contrast, no aberrant distribution of splenic T cells was observed by immunohistochemistry (Fig. 3F). Taken together, Rho GDI{alpha}beta deficiency correlates with defective intrathymic differentiation as well as defective T cell migration, especially exported from the thymus.

Elevation of eosinophil numbers in Rho GDI{alpha}beta–/– mice

In addition to defective B and T cell development, Rho GDI{alpha}beta–/– mice showed a characteristic feature. BM analyses showed an increase in eosinophil numbers in Rho GDI{alpha}beta–/– mice (Table II). We further observed that Mac1+Gr-1low myeloid cell population of the BM was increased specifically in Rho GDI{alpha}beta–/– mice (Fig. 4A). The Mac1+Gr-1– or the Mac1+Gr-1low population largely consists of immature macrophages and eosinophils, whereas the Mac1+Gr-1+ population mostly consists of neutrophils. Further flow cytometric analysis revealed an increase in the side scatter (SSC)-high population that was exclusively comprised of eosinophils (Fig. 4B). We further performed an assay for the histological detection of eosinophils and found that the eosinophil population was increased in the BM sections of Rho GDI{alpha}beta–/– mice compared with those of WT mice (Fig. 4, C, a–d). The eosinophil population was also increased in the lung of Rho GDI{alpha}beta–/– mice (Fig. 4, C, e and f) and inflammatory eyelid skin lesions (data not shown). We compared numbers of eosinophils in the BM and serum concentrations of IL-5 between the age-matched WT and Rho GDI{alpha}beta–/– mice groups (Fig. 4D). The average percentages of eosinophils (Rho GDI{alpha}beta–/–, 15%; WT, 5%) as well as the average serum concentrations of IL-5 (Rho GDI{alpha}beta–/–, 17 pg/ml; WT, 4.5 pg/ml) were significantly increased in Rho GDI{alpha}beta–/– mice, suggesting the contribution of defective T cell-mediated regulation leading to eosinophilia. The occurrence of eosinophilia associated with increased IL-5 tended to be age dependent because the differences were more clearly observed between the 12- to 55-wk-old groups than between the 6- and 11-wk-old groups (data not shown). These findings suggest the presence of hypersensitive disorders although functional correlations of eosinophilia to other phenotypes of Rho GDI{alpha}beta–/– mice remain to be elucidated.


Figure 4
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  		FIGURE 4. Increased eosinophil numbers in the BM and lung of Rho GDI{alpha}beta–/– mice. A, BM cells are prepared from WT and Rho GDI{alpha}beta–/– mice at 23 wk of age, and then analyzed for surface expression of the myeloid lineage markers, Mac-1 and Gr-1, by flow cytometry. B, The BM cell population indicated in A is further analyzed for SSC related to the cell granularity and forward scatter (FSC) related to the cell size by flow cytometry. C, BM sections from WT (a) and Rho GDI{alpha}beta–/– (b) mice at 32 wk of age are stained with H&E. BM sections from WT (c) and Rho GDI{alpha}beta–/– (d) mice at 32 wk of age are stained with the EoProbe kit, which is indicative of eosinophil-specific granules. Lung sections from WT (e) and Rho GDI{alpha}beta–/– (f) mice at 32 wk of age are stained with the EoProbe kit. Scale bars, 20 µm (a and b) and 50 µm (c–f). D, Relative eosinophil numbers in the BM and serum IL-5 concentrations are compared between WT and Rho GDI{alpha}beta–/– mice at 6–55 wk of age (WT, n = 35; Rho GDI{alpha}beta–/–, n = 37). *, p < 0.01; **, p < 0.001.

 
Defective in vitro proliferation and development of T cells in Rho GDI{alpha}beta–/– mice

The generation of mouse models for Rho family proteins, their regulators, and effectors have revealed the implication of Rho family proteins on proliferation, survival, and migration of B cell and T cell lineages (15, 27, 28), indicating Rho family proteins are critical components of multiple signaling pathways downstream of both Ag and chemokine receptors. To further determine whether Rho GDI{alpha}beta proteins are involved in lymphocyte proliferation and survival, we first examined DNA synthesis of lymphocytes obtained from Rho GDI{alpha}beta–/– mice using BrdU incorporation. Rho GDI{alpha}beta–/– splenic B cells displayed statistically significant increases in DNA synthesis in response to LPS compared with WT controls (Fig. 5A). In contrast, Rho GDI{alpha}beta–/– splenic T cells showed defective growth in vitro in response to anti-CD3 and anti-CD28 Abs (Fig. 5B). This finding was consistent with the lower levels of in vivo CD3 expression in Rho GDI{alpha}beta–/– T cells, suggesting the presence of improper T cell development. We next examined the effect of Rho GDI{alpha}beta deficiency on cell survival during intrathymic differentiation. However, apoptosis of Rho GDI{alpha}beta–/– thymocytes showed no significant change in the double-negative (DN), DP, CD4+ SP, and CD8+ SP populations as analyzed by flow cytometry using annexin V and propidium iodide (Fig. 5C). These findings indicate proliferation of T cells rather than B cells is impaired in Rho GDI{alpha}beta–/– mice, which presumably results in predominant effects on in vivo T cell development.

Responsiveness to chemokines of lymphocytes from Rho GDI{alpha}beta–/– mice

To address underlying mechanisms of the reduction of MZ B cells in the spleen as well as the retention of late medullary T cells in the thymus, we analyzed their chemotactic activities. Transwell migration assays showed slightly enhanced responsiveness of Rho GDI{alpha}beta–/– splenic B cells to the chemokines including CXCL12 (p < 0.05) (Fig. 5D). In contrast, splenic T cells were defective in response to CXCL12 (p < 0.01) and both CCL21 and CCL19 (p < 0.001) (Fig. 5E), but not to CXCL13 (data not shown). However, neither CD4+ SP nor CD8+ SP cells from the Rho GDI{alpha}beta–/– thymus showed significant change in similar chemotactic conditions (Fig. 5, F and G). Consistently, immune-fluorescent profiles showed chemokine receptor CCR7, a receptor for both CCL21 and CCL19, was down-regulated in splenic T cells (Fig. 5H) but not in CD4+ SP cells (Fig. 5I) and other T cell subpopulations in the thymus from Rho GDI{alpha}beta–/– mice (data not shown). These findings suggest the splenic T cells but not the thymocytes from Rho GDI{alpha}beta–/– mice were certainly defective in CCR7 signaling.

Reduced overall protein levels and partially sustained GTP-bound active states of Rho proteins in Rho GDI{alpha}beta–/– lymphocytes

To examine biochemical states of Rho GDI deficiencies in vivo, we first compared the relative protein levels and GTP-binding activities of RhoA, Rac1, and Cdc42 among lymphoid organs from Rho GDI{alpha}–/–, Rho GDIbeta–/–, and Rho GDI{alpha}beta–/– mice at 16 wk of age. Western blot analyses showed the levels of all three members were reduced to ~50% in the BM and to 70–30% in the spleen and thymus from these Rho GDI-deficient mice (Fig. 6, A and B, left panels). The reductions of Rho proteins in the spleen and thymus were more pronounced in Rho GDI{alpha}beta–/– mice than in Rho GDI{alpha}–/– or Rho GDIbeta–/– mice. Furthermore, the effect of Rho GDI deficiency appears to be age dependent because mice older than 16 wk of age reproducibly showed reduced protein levels of Rho family proteins. However, pull-down assays showed the residual part of RhoA, Rac1, and Cdc42 in Rho GDI{alpha}beta–/– BM cells tended to bind to GTP tightly (Fig. 6, A and B, right panels). The residual part of RhoA and Rac1 in Rho GDI{alpha}beta–/– splenocytes also tended to bind to GTP tightly although the levels of GTP-bound Cdc42 were less prominently elevated than those of GTP-bound RhoA and Rac1 (Fig. 6, A and B, right middle panels). These compensatory increases in RhoA, Rac1, and Cdc42 activities in the BM and spleen were not readily detected in the thymocytes from Rho GDI{alpha}beta–/– mice. We further analyzed the activities of Rho proteins in splenic B and T cell fractions and found the levels of GTP-bound active forms of RhoA and Rac1 were elevated in both of the fractions (Fig. 6C). In addition, mRNA expressions of Rho family proteins were basically at the similar levels between WT and Rho GDI{alpha}beta–/– mice (Fig. 6D) except for the RhoA expression in the spleen from Rho GDI{alpha}beta–/– mice, suggesting Rho GDI{alpha}beta deficiency has no obvious effects on transcription but may directly impair stability and activity of Rho family proteins. Thus, Rho family protein levels were generally decreased in the BM, spleen, and thymus whereas compensatory increases in Rho family activities were detected in the BM and spleen but not in the thymus from Rho GDI{alpha}beta–/– mice.


Figure 6
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  		FIGURE 6. Rho protein levels and activities in Rho GDI-deficient mice. A, Rho protein levels and activities in the BM, spleen, and thymus are compared among WT and Rho GDI-deficient mice as indicated. Protein levels of {alpha}-tubulin are shown as loading controls for Western blot analyses (left panels). After pull-down, these GTP-bound proteins are detected using corresponding Abs (middle panels). Loading controls are one-twentieth of the total proteins used for pull-down assays (right panels). Representative data from four independent experiments are shown here. B, Relative protein levels are calculated on the basis of results by Western blot and normalized to the WT values; GTP-bound forms (%) were determined by pull-down assays normalized to protein levels of loading controls. Results represent those obtained with four sets of mice of each phenotype at 16 wk of age. C, RhoA and Rac1 activities in the splenic B cells (left) and T cells (right) are compared between WT and Rho GDI{alpha}beta–/– mice as indicated. D, mRNA levels of RhoA, Rac1, and Cdc42 are analyzed by quantitative RT-PCR. Ratios normalized to the GAPDH controls are compared in splenocytes and thymocytes between WT and Rho GDI{alpha}beta–/– mice (n = 4) at 4–7 wk of age. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Rho GDI{alpha}beta deficiency possibly defines a new genetic syndrome of hemopoietic cells: defective positioning of MZ B cell compartment, defective export of mature T cells from the thymus, and specific phenotypes such as eosinophilia, lower levels of CD3 expression, and accumulation of B cells with reduction of T cells in the MLN. However, the complicated syndrome does not appear to be the direct result of a given mechanism but the consequence of several mechanisms involved in B and T cell development and trafficking. Positioning of B cells in the splenic MZ is reported to be a process that requires several factors (29): clonal signaling, lifespan and survival, retention, movement, and migration, transcription factors (NF-{kappa}B, RelB), receptor signaling (S1P1, Notch), and integrin-mediated adhesiveness. Directional T cell migration also requires integrin signaling that interacts with the actin cytoskeleton to gain locomotive force (30, 31, 32, 33), as well as chemokine signaling that polarizes T cell movement (31, 34, 35, 36, 37). Eosinophil numbers have reportedly been controlled at the level of the Th2 lymphocyte response to chemokines via CCR3, CCR4, and CCR8 (38) and the subsequent elaboration of IL-5, which exerts both direct and indirect effects on the inflammatory cells. Some biological aspects of the phenotype of Rho GDI{alpha}beta–/– mice could be directly attributed to our findings such as defective intrathymic T cell development, defective T cell chemokine signaling from CCR7, and elevated serum levels of IL-5 in Rho GDI{alpha}beta–/– mice. However, it seems difficult to elucidate individual mechanisms involved in combined Rho GDI{alpha}beta deficiency because Rho GDI{alpha}beta regulates several members of Rho family proteins simultaneously.

It is intriguing that mice are able to survive in the absence of major Rho GDI functions because Rho GDIs play critical roles for membrane-cytosol shuttling processes of Rho proteins in many cellular responses. Additionally, the phenotypic differences between WT and Rho GDI{alpha}beta–/– mice were age dependent, probably reflecting the chronic and complicated nature of Rho GDI{alpha}beta deficiency. Rho GDI{alpha}beta deficiency could cause two conceivable outcomes in vivo as follows: Rho GDI{alpha}beta deficiency induces activation of Rho proteins by making them available to another round of activation cycle, and Rho GDI{alpha}beta deficiency induces reduction of Rho protein levels by making them susceptible to proteolytic degradation. In fact, Smurf1 binds to nucleotide-free Rho and promotes Rho ubiquitination, leading to its proteolytic degradation (39), and caspase 3 mediates cleavage of Rac1 in apoptotic lymphoma cells (40). We observed an overall reduction in protein levels of RhoA, Rac1, and Cdc42 in the Rho GDI{alpha}beta–/– spleen and thymus, as well as prolonged activation of the residual part of RhoA, Rac1, and CDC42 mainly in the spleen. Rho GDI{alpha}beta–/– mice tended to develop defects of T cell function rather than those of B cell function, which may correlate with the compensation of Rho protein activities in the spleen but not in the thymus. Thus, the phenotype of Rho GDI{alpha}beta–/– mice appears to be caused primarily by the reduction in the total Rho protein levels, and this may be secondarily modified by sustained activation of the residual RhoA, Rac1, and Cdc42 proteins in a lymphoid organ-specific manner.

Rho is a critical mediator of TCR signaling required for thymocyte differentiation and survival in vivo. Lsc, a GTP exchange factor of Rho, is activated the G{alpha} (13) subunit of heterotrimeric G proteins and links the activation of G{alpha} (12) and G{alpha} (13)-coupled receptors to actin polymerization in B and T cells. Knockout studies showed Lsc is a critical regulator of MZ B cells and lymphocyte migration (41). Considering the similarity of the phenotype between mice lacking Rho GDI{alpha}beta and Lsc, part of their physiological function appears to overlap especially in regulating Rho activation. Importantly, Lsc–/– mice on a BALB/c background showed increased numbers of thymocytes and hyperplasia of the thymus, implicating Lsc in thromboxane (TXA) 2 signaling to T cells (42). Since TXA2 activates apoptosis of immature thymocytes, it is intriguing whether Rho GDI{alpha}beta–/– mice on a BALB/c background will develop hyperplasia of the thymus because of decreased TXA2-mediated apoptosis.

Rho GDI{alpha}beta deficiency caused considerably variable effects on lymphoid cells, which may be explained by a variety of reduced Rho protein levels and sustained activities as discussed. However, this may be explained by the multiplicity of target Rho proteins as well. We have not yet studied individual roles of Rho proteins, such as RhoA, RhoB, RhoC, RhoG, Cdc42, Rac1, Rac2, and Rac3, because Rho GDI{alpha}beta are general regulators for all these members. Therefore, our current notion is that Rho GDI{alpha}beta deficiency is likely to cause multiple, but usually partial, disruption of signaling pathways that involve more than eight members of Rho family proteins, leading to the complicated phenotype of Rho GDI{alpha}beta–/– mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Grant-In-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grants 14028074, 15024275, 16022268, and 17014090; to J.M.). Back

2 Address correspondence and reprint requests to Dr. Jun Miyoshi, Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Nakamichi 1-3-2, Higashinari-ku, Osaka 537-8511, Japan. E-mail address: miyosi-ju{at}mc.pref.osaka.jp Back

3 Abbreviations used in this paper: GDI, GDP-dissociation inhibitor; WT, wild type; BM, bone marrow; NF, newly formed; T1, transitional 1; T2, transitional 2; FO, follicular; MZ, marginal zone; MLN, mesenteric lymph node; DN, double negative; DP, double positive; SP, single positive; TXA, thromboxane A. Back

Received for publication March 16, 2006. Accepted for publication September 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Etienne-Manneville, S., A. Hall. 2002. Rho GTPases in cell biology. Nature 420: 629-635. [Medline]
  2. Wennerberg, K., C. J. Der. 2004. Rho-family GTPases: it’s not only Rac and Rho (and I like it). J. Cell Sci. 117: 1301-1312. [Abstract/Free Full Text]
  3. Erickson, J. W., R. A. Cerione. 2004. Structural elements, mechanism, and evolutionary convergence of Rho protein-guanine nucleotide exchange factor complexes. Biochemistry 43: 837-842. [Medline]
  4. Raftopoulou, M., A. Hall. 2004. Cell migration: Rho GTPases lead the way. Dev. Biol. 265: 23-32. [Medline]
  5. Yang, F. C., S. J. Atkinson, Y. Gu, J. B. Borneo, A. W. Roberts, Y. Zheng, J. Pennington, D. A. Williams. 2001. Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization. Proc. Natl. Acad. Sci. USA 98: 5614-5618. [Abstract/Free Full Text]
  6. Stramer, B., W. Wood, M. J. Galko, M. J. Redd, A. Jacinto, S. M. Parkhurst, P. Martin. 2005. Live imaging of wound inflammation in Drosophila embryos reveals key roles for small GTPases during in vivo cell migration. J. Cell Biol. 168: 567-573. [Abstract/Free Full Text]
  7. Decker, M., C. Moon, S. Le Bras. 2005. The immunological synapse and Rho GTPases. Curr. Top. Microbiol. Immunol. 291: 61-90. [Medline]
  8. Cyster, J. G.. 1992. Chemokines and cell migration in secondary lymphoid organs. Science 286: 2098-2102.
  9. Melchers, F., A. G. Rolink, C. Schaniel. 1999. The role of chemokines in regulating cell migration during humoral immune responses. Cell 99: 351-354. [Medline]
  10. Malorni, W., M. G. Quaranta, E. Straface, L. Falzano, A. Fabbri, M. Viora, C. Fiorentini. 2003. The Rac-activating toxin cytotoxic necrotizing factor 1 oversees NK cell-mediated activity by regulating the actin/microtubule interplay. J. Immunol. 171: 4195-4202. [Abstract/Free Full Text]
  11. Garcia-Bernal, D., N. Wright, E. Sotillo-Mallo, C. Nombela-Arrieta, J. V. Stein, X. R. Bustelo, J. Teixido. 2005. Vav1 and Rac control chemokine-promoted T lymphocyte adhesion mediated by the integrin {alpha}4beta1. Mol. Biol. Cell 16: 3223-3235. [Abstract/Free Full Text]
  12. Gottig, S., D. Mobest, B. Ruster, B. Grace, S. Winter, E. Seifried, J. Gille, T. Wieland, R. Henschler. 2006. Role of the monomeric GTPase Rho in hematopoietic progenitor cell migration and transplantation. Eur. J. Immunol. 36: 180-189. [Medline]
  13. Roberts, A. W., C. Kim, L. Zhen, J. B. Lowe, R. Kapur, B. Petryniak, A. Spaetti, J. D. Pollock, J. B. Borneo, G. B. Bradford, et al 1999. Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense. Immunity 10: 183-196. [Medline]
  14. Croker, B. A., D. M. Tarlinton, L. A. Cluse, A. J. Tuxen, A. Light, F. C. Yang, D. A. Williams, A. W. Roberts. 2002. The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-1a and marginal zone B lymphocytes. J. Immunol. 168: 3376-3386. [Abstract/Free Full Text]
  15. Walmsley, M. J., S. K. Ooi, L. F. Reynolds, S. H. Smith, S. Ruf, A. Mathiot, L. Vanes, D. A. Williams, M. P. Cancro, V. L. Tybulewicz. 2003. Critical roles for Rac1 and Rac2 GTPases in B cell development and signaling. Science 302: 459-462. [Abstract/Free Full Text]
  16. Benvenuti, F., S. Hugues, M. Walmsley, S. Ruf, L. Fetler, M. Popoff, V. L. Tybulewicz, S. Amigorena. 2004. Requirement of Rac1 and Rac2 expression by mature dendritic cells for T cell priming. Science 305: 1150-1153. [Abstract/Free Full Text]
  17. Wells, C. M., M. J. Walmsley, S. Ooi, V. Tybulewicz, A. J. Ridley. 2004. Rac1-deficient macrophages exhibit defects in cell spreading and membrane ruffling but not migration. J. Cell Sci. 117: 1259-1268. [Abstract/Free Full Text]
  18. Takai, Y., T. Sasaki, T. Matozaki. 2001. Small GTP-binding proteins. Physiol. Rev. 81: 259-270.
  19. Gandhi, P. N., R. M. Gibson, X. Tong, J. Miyoshi, Y. Takai, M. Konieczkowski, J. R. Sedor, A. L. Wilson-Delfosse. 2004. An activating mutant of Rac1 that fails to interact with Rho GDP-dissociation inhibitor stimulates membrane ruffling in mammalian cells. Biochem. J. 378: 409-419. [Medline]
  20. Gosser, Y. Q., T. K. Nomanbhoy, B. Aghazadeh, D. Manor, C. Combs, R. A. Cerione, M. K. Rosen. 1997. C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases. Nature 387: 814-819. [Medline]
  21. Hoffman, G. R., N. Nassar, R. A. Cerione. 2000. Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI. Cell 100: 345-356. [Medline]
  22. Scheffzek, K., I. Stephan, O. N. Jensen, D. Illenberger, P. Gierschik. 2000. The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI. Nat. Struct. Biol. 7: 122-126. [Medline]
  23. Golovanov, A. P., T. H. Chuang, C. DerMardirossian, I. Barsukov, D. Hawkins, R. Badii, G. M. Bokoch, L. H. Lian, G. C. K. Roberts. 2001. Structure-activity relationships in flexible protein domains: regulation of Rho GTPases by RhoGDI and D4 GDI. J. Mol. Biol. 305: 121-135. [Medline]
  24. Olofsson, B.. 1999. Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling. Cell. Signal. 11: 545-554. [Medline]
  25. Togawa, A., J. Miyoshi, H. Ishizaki, M. Tanaka, A. Takakura, H. Nishioka, H. Yoshida, T. Doi, A. Mizoguchi, N. Matsuura, et al 1999. Progressive impairment of kidneys and reproductive organs in mice lacking Rho GDI{alpha}. Oncogene 18: 5373-5380. [Medline]
  26. Yin, L., P. Schwartzberg, T. M. Scharton-Kersten, L. Staudt, M. Lenardo. 1997. Immune responses in mice deficient in Ly-GDI, a lymphoid-specific regulator of Rho GTPases. Mol. Immunol. 34: 481-491. [Medline]
  27. Fukui, Y., O. Hashimoto, T. Sanui, T. Oono, H. Koga, M. Abe, A. Inayoshi, M. Noda, M. Oike, T. Shirai, et al 2001. Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration. Nature 412: 826-831. [Medline]
  28. Takesono, A., R. Horai, M. Mandai, D. Dombroski, P. L. Schwartzberg. 2004. Requirement for Tec kinases in chemokine-induced migration and activation of Cdc42 and Rac. Curr. Biol. 14: 917-922. [Medline]
  29. Martin, F., J. F. Kearney. 2002. Marginal-zone B cells. Nat. Rev. Immunol. 2: 323-335. [Medline]
  30. Laudanna, C., J. Y. Kim, G. Constantin, E. Butcher. 2002. Rapid leukocyte integrin activation by chemokines. Immunol. Rev. 186: 37-46. [Medline]
  31. Petrie, H. T.. 2003. Cell migration and the control of post-natal T-cell lymphopoiesis in the thymus. Nat. Rev. Immunol. 3: 859-866. [Medline]
  32. Guo, W., F. G. Giancotti. 2004. Integrin signalling during tumour progression. Nat. Rev. Mol. Cell Biol. 5: 816-826. [Medline]
  33. Mitra, S. K., D. A. Hanson, D. D. Schlaepfer. 2005. Focal adhesion kinase: in command and control of cell motility. Nat. Rev. Mol. Cell Biol. 6: 56-68. [Medline]
  34. Chaffin, K. E., R. M. Perlmutter. 1991. A pertussis toxin-sensitive process controls thymocyte emigration. Eur. J. Immunol. 21: 2565-2573. [Medline]
  35. Ueno, T., K. Hara, M. S. Willis, M. A. Malin, U. E. Hopken, D. H. Gray, K. Matsushima, M. Lipp, T. A. Springer, R. L. Boyd, et al 2002. Role for CCR7 ligands in the emigration of newly generated T lymphocytes from the neonatal thymus. Immunity 16: 205-218. [Medline]
  36. Campbell, J. J., J. Pan, E. C. Butcher. 1999. Cutting edge: developmental switches in chemokine responses during T cell maturation. J. Immunol. 163: 2353-2357. [Abstract/Free Full Text]
  37. Misslitz, A., O. Pabst, G. Hintzen, L. Ohl, E. Kremmer, H. T. Petrie, R. Forster. 2004. Thymic T cell development and progenitor localization depend on CCR7. J. Exp. Med. 200: 481-491. [Abstract/Free Full Text]
  38. Zlotnik, A., O. Yoshie. 2000. Chemokines: a new classification system and their role in immunity. Immunity 12: 121-127. [Medline]
  39. Wang, H. R., Y. Zhang, B. Ozdamar, A. A. Ogunjimi, E. Alexandrova, G. H. Thomsen, J. L. Wrana. 2003. Regulation of cell polarity and protrusion formation by targeting RhoA for degradation. Science 302: 1775-1779. [Abstract/Free Full Text]
  40. Zhang, B., Y. Zhang, E. Shacter. 2003. Caspase 3-mediated inactivation of rac GTPases promotes drug-induced apoptosis in human lymphoma cells. Mol. Cell. Biol. 23: 5716-5725. [Abstract/Free Full Text]
  41. Girkontaite, I., K. Missy, V. Sakk, A. Harenberg, K. Tedford, T. Potzel, K. Pfeffer, K. D. Fischer. 2001. Lsc is required for marginal zone B cells, regulation of lymphocyte motility and immune responses. Nat. Immunol. 2: 855-862. [Medline]
  42. Harenberg, A., I. Girkontaite, K. Giehl, K. D. Fischer. 2005. The Lsc RhoGEF mediates signaling from thromboxane A2 to actin polymerization and apoptosis in thymocytes. Eur. J. Immunol. 35: 1977-1986. [Medline]



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