Impaired T lymphocyte trafficking in mice deficient in an actin-nucleating protein, mDia1

Abstract

Trafficking of immune cells is controlled by directed migration of relevant cells toward chemotactic signals. Actin cytoskeleton undergoes continuous remodeling and serves as machinery for cell migration. The mDia family of formins and the Wiskott-Aldrich syndrome protein (WASP)-Arp2/3 system are two major actin nucleating-polymerizing systems in mammalian cells, with the former producing long straight actin filaments and the latter producing branched actin meshwork. Although much is known about the latter, the physiological functions of mDia proteins are unclear. We generated mice deficient in one mDia isoform, mDia1. Although mDia1(-/-) mice were born and developed without apparent abnormality, mDia1(-/-) T lymphocytes exhibited impaired trafficking to secondary lymphoid organs in vivo and showed reduced chemotaxis, little actin filament formation, and impaired polarity in response to chemotactic stimuli in vitro. Similarly, mDia1(-/-) thymocytes showed reduced chemotaxis and impaired egression from the thymus. These results suggest that mDia1 plays a distinct role in chemotaxis in T lymphocyte trafficking.

Figure 1.

Decreased number of T cells in secondary lymphoid organs of mDia1^−/− mice. (A) Western blot analysis. The homogenates of the brain, lung, and thymus of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice were subjected to immunoblot analysis for mDia1, mDia2, mDia3, and β-actin (B) Weight of the body and lymphoid organs of wild-type (shaded bars) and mDia1^−/− (open bars) mice (n = 3 for each group). The experiment was performed twice with reproducible results. (C) Immunohistochemistry of the spleen and lymph node. The spleen and lymph node of mDia1^+/+ and mDia1^−/− mice were stained for Thy1.2 (green) and B220 (red). Representative observation from samples of two mice. Bar, 100 μm. (D) Cell population analysis in the spleen, lymph node, and blood. The numbers of total cells, Thy1.2⁺ T cells, B220⁺ B cells, CD4⁺ T cells, CD8⁺ T cells, CD11c⁺ cells, and CD11b⁺ cells in the spleen, lymph node, and blood were determined in mDia1^+/+ (shaded bars) and mDia1^−/− (open bars) mice. (E) Cell population analysis in the thymus. The numbers of total cells and indicated subsets of thymocytes (DN, CD4⁻CD8⁻; DP, CD4⁺CD8⁺; CD4-SP, CD4⁺CD8⁻; CD8-SP, CD4⁻CD8⁺) in the thymus were determined in mDia1^+/+ (shaded bars) and mDia1^−/− (open bars) mice. The bottom panel shows the numbers of CD69^loCD62L^hiCD4-SP and CD8-SP cells in the thymus of mDia1^+/+ and mDia1^−/− mice (n = 4 for each group). Unless otherwise stated, experiments shown in D and E were performed using three mice for each group more than twice with similar results, and results from one experiment are shown. All data are shown as means ± SEM. *, P < 0.05 versus the number of the corresponding population in mDia1^+/+ wild-type mice. The axillary and inguinal lymph nodes were used as peripheral lymph nodes for analysis in these experiments.

Figure 2.

Impaired migration of mDia1^−/− T cells and thymocytes. (A and B) Impaired chemotaxis toward chemokines in vitro. Migration of T cells and B cells (A; n = 4 for each groups) and thymocytes (B; n = 3 for each group) from mDia1^+/+ (shaded bars) and mDia1^−/− (open bars) mice toward the indicated chemokines was examined using a transwell chamber. In B, populations of thymocytes that migrated to the bottom chamber were analyzed by flow cytometry after staining for CD4 and CD8. *, P < 0.05. (C) Impaired thymocyte egression from the thymus in organ culture. Thymocytes that egressed from the thymic lobe toward CCL21 were stained for CD4 and CD8 and analyzed by flow cytometry (n = 3 for each group). *, P < 0.05. (D) Adoptive transfer experiment. T cells were isolated from the spleen of mDia1^+/+ and mDia1^−/− mice and were labeled with different fluorescent dyes, administered to wild-type C57BL/6 mice or mDia1^−/− recipient mice (n = 3 for each group). The ratios of the two populations in the blood and migrating to the spleen and the axillary and inguinal lymph nodes were analyzed after 2 h. All data are shown as means ± SEM.

Figure 3.

Impaired chemokine-induced actin polymerization and polarization of mDia1^−/− T cells. (A) F-actin production to chemokine stimulation. T cells from mDia1^+/+ (blue line) and mDia1^−/− (red line) mice were stimulated with two concentrations of CCL21 for the indicated times, stained with Oregon green–phalloidin, and analyzed by flow cytometry. Mean fluorescence intensity of the entire cell population was determined for each group and is shown with that of unstimulated mDia1^+/+ cells as 100%. (B) Impaired chemokine responses of mDia1^−/− T cells. T cells from mDia1^+/+ and mDia1^−/− mice were stimulated with or without CCL21 and stained for talin, F-actin, and DNA, as indicated. Bar, 10 μm.

Figure 4.

Impaired immune responses of mDia1^−/− T cells. (A and B) Impaired proliferation of mDia1^−/− T cell. T cells from mDia1^+/+ (shaded bars) and mDia1^−/− (open bars) mice were cultured with plate-bound anti-CD3 antibody for 48 h (A) or with CD11c⁺ dendritic cells for 72 h (B). Cell proliferation was determined by [³H]thymidine uptake. (C) Impaired contact hypersensitivity of mDia1^−/− mice. mDia1^+/+ (shaded bars) and mDia1^−/− (open bars) mice were subjected to the DNFB-induced contact hypersensitivity, and ear thickness was measured. All data are shown as means ± SEM.

Figure 5.

Analysis on signaling molecules. (A) Flow cytometry for chemokine receptor expression. mDia1^+/+ and mDia1^−/− T cells were stained with anti-CXCR4 or anti-CCR7 antibody (continuous line) or control antibody (dotted line) and analyzed by flow cytometry. (B) Western blot analysis of lysates of mDia1^−/− T cells. T cells were prepared from mDia1^+/+ and mDia1^−/− mice and subjected to immunoblot analysis for mDia1, mDia2, mDia3, RhoA, Rac1, Cdc42, ROCK-1, ROCK-2, WASP, or Arp2/3 subunit 34. The amount of WASP in mDia1^−/− T cells was examined by serial dilutions of the mDia1^+/+ lysates. (C) Degradation of WASP. T cells were treated with 10 μg/ml cycloheximide (CHX) for the indicated times, and cell lysates were subjected to immunoblot analysis with antibody to WASP (top). For comparison, two-fold amounts of lysates and longer color development were used for analysis of mDia1^−/− T cells. The density of each band on the immunoblots was quantified by the densitometry and plotted (mDia1^+/+, blue line; mDia1^−/−, pink line) against incubation time with the density at 0 h as 100% (right). (D) Effects of proteasome inhibitor. T cells were treated with the indicated concentrations of MG132 for 4 h, and cell lysates were subjected to immunoblot for WASP. (E) Overexpression of WASP in mDia1^−/− T cells. Wild-type or mDia1^−/− T cells were transfected with pEGFP-WASP or pEGFP. Expression of EGFP-WASP was confirmed by Western blotting and flow cytometry (top left and top middle). Densitometry of the immunoblot of the cells transfected with 4 μg pEGFP-WASP showed that EGFP-WASP was expressed at 17.4 and 18.8% of the endogenous WASP in wild-type and mDia1^−/− cells, respectively. Wild-type and mDia1^−/− T cells transfected with 4 μg of pEGFP or pEGFP-WASP were subjected to the transwell migration assay (top right) or fluorescence microscopy after staining with Texas red–phalloidin (bottom left), followed by measurement of the fluorescence intensity of F-actin staining (bottom right; n = 30 cells each). Bar, 10 μm.