Defective Chemokine-Directed Lymphocyte Migration and Development in the Absence of Rho Guanosine Diphosphate-Dissociation Inhibitors α and β

Generation of Rho GDIαβ^−/− mice

Rho GDIα^−/− mice were null mutants as previously described (25). Rho GDIβ^−/− mice were newly generated by replacing the 1.7-kb DNA region covering the promoter region and the exons 1–2 of the Rho GDIβ 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 GDIβ gene were identified by Southern hybridization using the 5′ and 3′ probes as indicated (see Fig. 1⇓A), 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 GDIα, 5′-AGAACAGGAACCCACTGCTGAGCA-3′ and 5′-AGTGCTGCATACCAAGGTCAGTCG-3′; Rho GDIβ, 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 GDIα allele (25) and a 107-bp band for the WT Rho GDIβ 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.

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FIGURE 1.

Impaired spermatogenesis of Rho GDI-deficient mice. A, Targeting strategy generating Rho GDIβ^−/− 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 GDIα and Rho GDIβ 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 GDIα, Rho GDIβ, and Rho GDIαβ. 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 GDIβ, Rho GDIα, and Rho GDIαβ.

Immunoblot analysis

Cell extracts were prepared as described (25). The following Abs were used for immunoblot analysis: anti-RhoA, anti-Cdc42, and anti-α-tubulin Abs were obtained from Santa Cruz Biotechnology, anti-Rac1 Abs were obtained from BD Transduction Laboratories, and anti-β-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 × 10⁵ cells/ml for B cells and at 2.5 × 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 × 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 × 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.

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 × 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.

General properties of Rho GDIαβ^−/− mice

Rho GDIαβ^−/− double-null mice were derived from Rho GDIα^−/− mice (25) and Rho GDIβ^−/− mice that were newly generated (Fig. 1⇑, A and B). Importantly, targeted disruption of lymphoid-specific Rho GDIβ resulted in neither age-dependent degeneration of kidney function nor male infertility as observed with Rho GDIα^−/− mice (25). The specific disruptions of Rho GDIα and Rho GDIβ genes were confirmed by Western blot analyses using extracts from splenic B cells and T cells (Fig. 1⇑C). Disrupting Rho GDIα or Rho GDIβ isoform did not alter the protein levels of its counterpart, Rho GDIβ or Rho GDIα, respectively, suggesting that there was no compensation between Rho GDIα and Rho GDIβ protein levels in the lymphocytes. Rho GDIαβ^−/− mice were exclusively obtained by in vitro or in vivo fertilization and oviduct transfer using Rho GDIα^+/− mice on the Rho GDIβ^−/− background because natural mating of Rho GDIα^+/− Rho GDIβ^−/− mouse pairs yielded no sibling (Table I⇓). Approximately 6.5% of pups were identified as Rho GDIαβ^−/− mice. Although nearly 75% of Rho GDIαβ^−/− 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 GDIαβ^−/− mice were totally infertile; histologically, Rho GDIαβ^−/− testes showed drastic testicular degeneration (Fig. 1⇑D) compared with Rho GDIα^−/− and Rho GDIβ^−/− testes. Therefore, Rho GDIα and Rho GDIβ play synergistic roles in maintaining the male reproductive function.

Aberrant distribution of B cell population in Rho GDIαβ^−/− mice

Since expression of Rho GDIα and Rho GDIβ overlaps in the hemopoietic cell lineages, we first analyzed the number and distribution of B cells in Rho GDIα^−/−, Rho GDIβ^−/−, and Rho GDIαβ^−/− 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 GDIαβ^−/− mice (p < 0.05, data not shown). Analysis of BM from Rho GDIαβ^−/− 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 GDIαβ^−/− 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 GDIαβ^−/− BM at 12–32 wk of age (Table II⇓).

B cell numbers of the spleen were reduced by 40% in Rho GDIαβ^−/− 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 GDIαβ^−/− mice into populations of newly formed (NF; B220⁺CD23⁻CD21⁻), transitional 1 (T1; CD23⁻CD21^lowIgM^highIgD^low), transitional 2 (T2; CD23⁺CD21^highIgM^highIgD^high), follicular (FO; CD23⁺CD21^intIgM^lowIgD^high), and MZ (CD23⁻CD21^highIgM^highIgD^low) cells. These analyses revealed that subsets of MZ B cells were drastically reduced in Rho GDIαβ^−/− mice compared with those of WT mice at 12 wk of age (Table II⇑ and Fig. 2⇓A), whereas NF, T1, T2, and FO cells were moderately reduced by 20–30% in Rho GDIαβ^−/− mice. This near absence of the MZ B cell population was confirmed by immunohistochemistry (Fig. 2⇓B); the localization of B cells was disorganized in the border region of the white pulp of the Rho GDIαβ^−/− spleen. The distribution of MOMA-1-positive metallophilic MZ macrophages appeared to be unimpaired in Rho GDIαβ^−/− 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.

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FIGURE 2.

Reduction of splenic MZ B cells and accumulation of MLN B cells from Rho GDIαβ^−/− mice. A, Expression of CD21/CD35 and CD23 on B220⁺ splenic B cells from WT and Rho GDIαβ^−/− 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 GDIαβ^−/− mice (right). C, Lymphocytes of MLN from WT and Rho GDIαβ^−/− 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 GDIαβ^−/− mice compared with WT mice, while B cell proportion is increased in Rho GDIαβ^−/− mice.

In contrast, B cell numbers in the mesenteric lymph nodes (MLN) were increased by 2- to 3-fold in Rho GDIαβ^−/− mice (Table II⇑ and Fig. 2⇑C). 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 GDIαβ proteins differentially regulated the B cell populations in secondary lymphoid organs.

Aberrant distribution of T cell population in Rho GDIαβ^−/− 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 GDIα^−/−, Rho GDIβ^−/−, and Rho GDIαβ^−/− mice, respectively, when compared with WT controls (Fig. 3⇓A). 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 GDIαβ^−/− mice (Table II⇑). We then analyzed CD3⁺ cells in the spleen by surface expression of CD11a and CD44. The population of CD11a⁺CD44^low cells was reduced to 14.8% in Rho GDIαβ^−/− mice, indicating loss of naive T cells (Fig. 3⇓B). The numbers of CD3⁺ cells were also decreased in the MLN of Rho GDIαβ^−/− mice (Fig. 2⇑C). However, the total numbers of thymocytes in Rho GDIαβ^−/− mice were comparable to those of WT mice, and the size and weight of the thymus were not different between WT and Rho GDIαβ^−/− 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 GDIαβ^−/− mice (Table II⇑ and Fig. 3⇓C). Characteristically, T cell populations in Rho GDIαβ^−/− mice showed lower CD3 levels in peripheral blood (Fig. 3⇓A) and all the lymphoid organs examined, as representatively shown in the MLN (Fig. 2⇑C). 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 GDIαβ^−/− mice have insufficient capabilities for intrathymic development.

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FIGURE 3.

Defective T cell development and accumulation of CD4⁺ SP cells in the thymic medulla of Rho GDIαβ^−/− 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⁺CD44^low). C, Increases in CD4⁺ SP and CD8⁺ SP cells from Rho GDIαβ^−/− 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 GDIαβ^−/− 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 GDIαβ^−/− 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 × 10⁶ (WT) and 5.6 × 10⁶ (Rho GDIαβ^−/−). Those of LM cells are 2.7 × 10⁶ (WT) and 12.0 × 10⁶ (Rho GDIαβ^−/−). F, Immunohistochemistry of spleen sections stained with anti-CD4 (red) and anti-CD8 (green) Abs. Cryosections are prepared from WT and Rho GDIαβ^−/− mice at 12 wk of age. Scale bars, 200 μm.

We next addressed intrathymic distribution of T cells in the Rho GDIαβ^−/− mice. Immune fluorescence showed CD4⁺CD8⁺ double-positive (DP) cells were evenly distributed in the inner and outer cortex of the thymus (Fig. 3⇑D), consistent with the restored CD4⁺CD8⁺ DP cell population (Fig. 3⇑C). It is of note, however, that mature T cells, mostly comprised of CD4⁺ SP cells, accumulated in the medulla of the Rho GDIαβ^−/− thymus, whereas WT CD4⁺ SP cells seemed to constantly emigrate from the medulla toward peripheral blood (Fig. 3⇑D). 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⁻CD62L^high) rather than early medullary cells (CD4⁺CD69⁺CD62L^low) (Fig. 3⇑E). In addition, the CD4⁺CD69⁻CD62L^low 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. 3⇑F). Taken together, Rho GDIαβ deficiency correlates with defective intrathymic differentiation as well as defective T cell migration, especially exported from the thymus.

Elevation of eosinophil numbers in Rho GDIαβ^−/− mice

In addition to defective B and T cell development, Rho GDIαβ^−/− mice showed a characteristic feature. BM analyses showed an increase in eosinophil numbers in Rho GDIαβ^−/− mice (Table II⇑). We further observed that Mac1⁺Gr-1^low myeloid cell population of the BM was increased specifically in Rho GDIαβ^−/− mice (Fig. 4⇓A). The Mac1⁺Gr-1⁻ or the Mac1⁺Gr-1^low 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. 4⇓B). 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 GDIαβ^−/− mice compared with those of WT mice (Fig. 4⇓, C, a–d). The eosinophil population was also increased in the lung of Rho GDIαβ^−/− 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 GDIαβ^−/− mice groups (Fig. 4⇓D). The average percentages of eosinophils (Rho GDIαβ^−/−, 15%; WT, 5%) as well as the average serum concentrations of IL-5 (Rho GDIαβ^−/−, 17 pg/ml; WT, 4.5 pg/ml) were significantly increased in Rho GDIαβ^−/− 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 GDIαβ^−/− mice remain to be elucidated.

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FIGURE 4.

Increased eosinophil numbers in the BM and lung of Rho GDIαβ^−/− mice. A, BM cells are prepared from WT and Rho GDIαβ^−/− 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 GDIαβ^−/− (b) mice at 32 wk of age are stained with H&E. BM sections from WT (c) and Rho GDIαβ^−/− (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 GDIαβ^−/− (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 GDIαβ^−/− mice at 6–55 wk of age (WT, n = 35; Rho GDIαβ^−/−, n = 37). ∗, p < 0.01; ∗∗, p < 0.001.

Defective in vitro proliferation and development of T cells in Rho GDIαβ^−/− 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 GDIαβ proteins are involved in lymphocyte proliferation and survival, we first examined DNA synthesis of lymphocytes obtained from Rho GDIαβ^−/− mice using BrdU incorporation. Rho GDIαβ^−/− splenic B cells displayed statistically significant increases in DNA synthesis in response to LPS compared with WT controls (Fig. 5⇓A). In contrast, Rho GDIαβ^−/− splenic T cells showed defective growth in vitro in response to anti-CD3 and anti-CD28 Abs (Fig. 5⇓B). This finding was consistent with the lower levels of in vivo CD3 expression in Rho GDIαβ^−/− T cells, suggesting the presence of improper T cell development. We next examined the effect of Rho GDIαβ deficiency on cell survival during intrathymic differentiation. However, apoptosis of Rho GDIαβ^−/− 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. 5⇓C). These findings indicate proliferation of T cells rather than B cells is impaired in Rho GDIαβ^−/− mice, which presumably results in predominant effects on in vivo T cell development.

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FIGURE 5.

Effect of Rho GDIαβ deficiency on in vitro proliferation of splenic B and T cells, apoptosis of thymocytes, and migration activities of Rho GDIαβ^−/− lymphocytes toward chemokines. A, The frequency of BrdU-positive B cells from the spleen of WT (▪) and Rho GDIαβ^−/− mice (□) 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 GDIαβ^−/− 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 GDIαβ^−/− 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 GDIαβ^−/− 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 GDIαβ^−/− mice at 7–9 wk of age are used in Transwell chemotaxis assays. T cells from Rho GDIαβ^−/− 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 GDIαβ^−/− mice (n = 6) at 4–7 wk of age.

Responsiveness to chemokines of lymphocytes from Rho GDIαβ^−/− 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 GDIαβ^−/− splenic B cells to the chemokines including CXCL12 (p < 0.05) (Fig. 5⇑D). In contrast, splenic T cells were defective in response to CXCL12 (p < 0.01) and both CCL21 and CCL19 (p < 0.001) (Fig. 5⇑E), but not to CXCL13 (data not shown). However, neither CD4⁺ SP nor CD8⁺ SP cells from the Rho GDIαβ^−/− 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. 5⇑H) but not in CD4⁺ SP cells (Fig. 5⇑I) and other T cell subpopulations in the thymus from Rho GDIαβ^−/− mice (data not shown). These findings suggest the splenic T cells but not the thymocytes from Rho GDIαβ^−/− mice were certainly defective in CCR7 signaling.

Reduced overall protein levels and partially sustained GTP-bound active states of Rho proteins in Rho GDIαβ^−/− 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 GDIα^−/−, Rho GDIβ^−/−, and Rho GDIαβ^−/− 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 GDIαβ^−/− mice than in Rho GDIα^−/− or Rho GDIβ^−/− 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 GDIαβ^−/− BM cells tended to bind to GTP tightly (Fig. 6⇓, A and B, right panels). The residual part of RhoA and Rac1 in Rho GDIαβ^−/− 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 GDIαβ^−/− 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. 6⇓C). In addition, mRNA expressions of Rho family proteins were basically at the similar levels between WT and Rho GDIαβ^−/− mice (Fig. 6⇓D) except for the RhoA expression in the spleen from Rho GDIαβ^−/− mice, suggesting Rho GDIαβ 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 GDIαβ^−/− mice.

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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 α-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 GDIαβ^−/− 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 GDIαβ^−/− mice (n = 4) at 4–7 wk of age. ∗, p < 0.05.