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

Sakata D, Taniguchi H, Yasuda S, Adachi-Morishima A, Hamazaki Y, Nakayama R, Miki T, Minato N, Narumiya S - J. Exp. Med. (2007)

Bottom Line: Although much is known about the latter, the physiological functions of mDia proteins are unclear.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology,Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan.

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.

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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.
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fig2: 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.

Mentions: Given that trafficking of thymocytes and T cells is achieved by their migration to chemokines (1), we examined the response of these cells toward chemokines in vitro. The transwell assay revealed impaired chemotaxis of mDia1−/− T cells toward CXC chemokine ligand (CXCL) 12 and CC chemokine ligand (CCL) 21, the two chemokines implicated in in vivo trafficking to secondary lymphoid organs (13). On the other hand, no significant impairment was found in chemotaxis of mDia1−/− B cells toward CXCL12 (Fig. 2 A). Similarly, the transwell assay revealed impaired chemotaxis of CD4+CD8+, CD4+CD8−, and CD4−CD8+ thymocytes toward CXCL12 (14) and CCL21 in the mDia1−/− genotype (Fig. 2 B). To assess egression more directly, we used organ culture of the thymus. Egression of CD4+CD8− and CD4−CD8+ thymocytes from thymic lobes in response to CCL21 was significantly decreased in mDia1 deficiency (Fig. 2 C). These results indicate that impaired chemotaxis of thymocytes and T cells underlies the phenotype observed in mDia1−/− mice. However, T cell trafficking to lymphoid organs in vivo may not be determined solely by the migratory activity of T cells but can be influenced by other factors, including the adhesive property of high endothelium venules in the lymphoid organs (15). To examine this issue, we performed an adoptive transfer experiment, in which we labeled T cells from wild-type and mDia1−/− mice with different fluorescent dyes and infused them i.v. into wild-type or mDia1−/− recipients. The numbers of transferred cells that migrated into the spleen and lymph nodes were determined 2 h after the infusion. We observed that, compared with wild-type cells, only ∼50% of mDia1−/− T cells migrated into the spleen and lymph nodes and that the extent of the reduction was not different in wild-type and mDia1−/− recipients (Fig. 2 D). Consistently, the higher numbers of mDia1−/− cells remained in the circulation.


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

Sakata D, Taniguchi H, Yasuda S, Adachi-Morishima A, Hamazaki Y, Nakayama R, Miki T, Minato N, Narumiya S - J. Exp. Med. (2007)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2118705&req=5

fig2: 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.
Mentions: Given that trafficking of thymocytes and T cells is achieved by their migration to chemokines (1), we examined the response of these cells toward chemokines in vitro. The transwell assay revealed impaired chemotaxis of mDia1−/− T cells toward CXC chemokine ligand (CXCL) 12 and CC chemokine ligand (CCL) 21, the two chemokines implicated in in vivo trafficking to secondary lymphoid organs (13). On the other hand, no significant impairment was found in chemotaxis of mDia1−/− B cells toward CXCL12 (Fig. 2 A). Similarly, the transwell assay revealed impaired chemotaxis of CD4+CD8+, CD4+CD8−, and CD4−CD8+ thymocytes toward CXCL12 (14) and CCL21 in the mDia1−/− genotype (Fig. 2 B). To assess egression more directly, we used organ culture of the thymus. Egression of CD4+CD8− and CD4−CD8+ thymocytes from thymic lobes in response to CCL21 was significantly decreased in mDia1 deficiency (Fig. 2 C). These results indicate that impaired chemotaxis of thymocytes and T cells underlies the phenotype observed in mDia1−/− mice. However, T cell trafficking to lymphoid organs in vivo may not be determined solely by the migratory activity of T cells but can be influenced by other factors, including the adhesive property of high endothelium venules in the lymphoid organs (15). To examine this issue, we performed an adoptive transfer experiment, in which we labeled T cells from wild-type and mDia1−/− mice with different fluorescent dyes and infused them i.v. into wild-type or mDia1−/− recipients. The numbers of transferred cells that migrated into the spleen and lymph nodes were determined 2 h after the infusion. We observed that, compared with wild-type cells, only ∼50% of mDia1−/− T cells migrated into the spleen and lymph nodes and that the extent of the reduction was not different in wild-type and mDia1−/− recipients (Fig. 2 D). Consistently, the higher numbers of mDia1−/− cells remained in the circulation.

Bottom Line: Although much is known about the latter, the physiological functions of mDia proteins are unclear.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology,Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan.

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.

Show MeSH
Related in: MedlinePlus