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A point mutation in the murine Hem1 gene reveals an essential role for Hematopoietic protein 1 in lymphopoiesis and innate immunity.

Park H, Staehling-Hampton K, Appleby MW, Brunkow ME, Habib T, Zhang Y, Ramsdell F, Liggitt HD, Freie B, Tsang M, Carlson G, Friend S, Frevert C, Iritani BM - J. Exp. Med. (2008)

Bottom Line: Using a chemical mutagenesis strategy in mice to identify novel genes involved in immune cell functions, we positionally cloned a nonsense mutation in the Hem1 gene.Remarkably, some Rac-dependent functions, such as Th1 differentiation and nuclear factor kappaB (NF-kappaB)-dependent transcription of proinflammatory cytokines proceed normally in Hem1-deficient mice, whereas the production of Th17 cells are enhanced.These results demonstrate that Hem1 is essential for hematopoietic cell development, function, and homeostasis by controlling a distinct pathway leading to cytoskeletal reorganization, whereas NF-kappaB-dependent transcription proceeds independently of Hem1 and F-actin polymerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
Hem1 (Hematopoietic protein 1) is a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins. Orthologues of Hem1 in Dictyostelium discoideum, Drosophila melanogaster, and Caenorhabditis elegans are essential for cytoskeletal reorganization, embryonic cell migration, and morphogenesis. However, the in vivo functions of mammalian Hem1 are not known. Using a chemical mutagenesis strategy in mice to identify novel genes involved in immune cell functions, we positionally cloned a nonsense mutation in the Hem1 gene. Hem1 deficiency results in defective F-actin polymerization and actin capping in lymphocytes and neutrophils caused by loss of the Rac-controlled actin-regulatory WAVE protein complex. T cell development is disrupted in Hem1-deficient mice at the CD4(-)CD8(-) (double negative) to CD4(+)CD8(+) (double positive) cell stages, whereas T cell activation and adhesion are impaired. Hem1-deficient neutrophils fail to migrate in response to chemotactic agents and are deficient in their ability to phagocytose bacteria. Remarkably, some Rac-dependent functions, such as Th1 differentiation and nuclear factor kappaB (NF-kappaB)-dependent transcription of proinflammatory cytokines proceed normally in Hem1-deficient mice, whereas the production of Th17 cells are enhanced. These results demonstrate that Hem1 is essential for hematopoietic cell development, function, and homeostasis by controlling a distinct pathway leading to cytoskeletal reorganization, whereas NF-kappaB-dependent transcription proceeds independently of Hem1 and F-actin polymerization.

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T cell development is severely impaired in Hem1-deficient mice. Total thymocytes (A) and splenocytes (B) from Hem1−/− and WT littermate mice were stained with fluorescent-conjugated α-CD4, α-CD8, α-CD3ε, α-CD25, α-CD44, α-B220, α-GR1, α–TCR-γδ, α–TCR-β, and intracellular α-Foxp3 and analyzed by flow cytometry. Representative histograms of eight mice per group of cells that fall within a forward scatter (FSC) and side scatter (SSC) lymphocyte gate are shown. Total thymocyte and splenocyte number were multiplied by the percentage of cells that fell within each quadrant to obtain the total number of cells within each developmental subset (right). Cells negative for B220, GR1, CD4, and CD8 were analyzed with CD44 and CD25 staining to determine the DN1-DN4 fractions. Bars represent the mean ± SEM from eight mice. P-values are shown. The high side scatter cells in Hem1−/− spleens are myeloid and progenitors cells.
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fig4: T cell development is severely impaired in Hem1-deficient mice. Total thymocytes (A) and splenocytes (B) from Hem1−/− and WT littermate mice were stained with fluorescent-conjugated α-CD4, α-CD8, α-CD3ε, α-CD25, α-CD44, α-B220, α-GR1, α–TCR-γδ, α–TCR-β, and intracellular α-Foxp3 and analyzed by flow cytometry. Representative histograms of eight mice per group of cells that fall within a forward scatter (FSC) and side scatter (SSC) lymphocyte gate are shown. Total thymocyte and splenocyte number were multiplied by the percentage of cells that fell within each quadrant to obtain the total number of cells within each developmental subset (right). Cells negative for B220, GR1, CD4, and CD8 were analyzed with CD44 and CD25 staining to determine the DN1-DN4 fractions. Bars represent the mean ± SEM from eight mice. P-values are shown. The high side scatter cells in Hem1−/− spleens are myeloid and progenitors cells.

Mentions: Because Hem1-deficient mice are lymphopenic, we examined whether T and/or B cell development are impaired. The development of lymphocytes can be broken down into many distinct stages based on the presence or absence of particular intracellular and surface proteins, as determined by flow cytometry (29, 30). As shown in Fig. 4 A, Hem1−/− mice have a 30-fold mean reduction in thymocyte number with an impairment in αβ T cell development within the CD4−CD8− double-negative (DN) to CD4+CD8+ double-positive (DP) cell transition at the CD25midCD44− (DN3) to CD25−CD44−(DN4) cell stage, which is mediated by formation of the preTCR. The number of γδ thymocytes is also decreased, albeit to a lesser extent than αβ T cells (Fig. 4, A and B). The total numbers of peripheral CD4, CD8, and γδ T cells (Fig. 4 B) and B lymphocytes (not depicted) are reduced by at least 75%. The ratio of Foxp3+CD4+ T regulatory cells (Treg) to Foxp3−CD4+ T cells is significantly higher in both thymus (Fig. 4 A) and spleen (Fig. 4 B), suggesting that Hem1 regulates both lymphocyte development and homeostasis.


A point mutation in the murine Hem1 gene reveals an essential role for Hematopoietic protein 1 in lymphopoiesis and innate immunity.

Park H, Staehling-Hampton K, Appleby MW, Brunkow ME, Habib T, Zhang Y, Ramsdell F, Liggitt HD, Freie B, Tsang M, Carlson G, Friend S, Frevert C, Iritani BM - J. Exp. Med. (2008)

T cell development is severely impaired in Hem1-deficient mice. Total thymocytes (A) and splenocytes (B) from Hem1−/− and WT littermate mice were stained with fluorescent-conjugated α-CD4, α-CD8, α-CD3ε, α-CD25, α-CD44, α-B220, α-GR1, α–TCR-γδ, α–TCR-β, and intracellular α-Foxp3 and analyzed by flow cytometry. Representative histograms of eight mice per group of cells that fall within a forward scatter (FSC) and side scatter (SSC) lymphocyte gate are shown. Total thymocyte and splenocyte number were multiplied by the percentage of cells that fell within each quadrant to obtain the total number of cells within each developmental subset (right). Cells negative for B220, GR1, CD4, and CD8 were analyzed with CD44 and CD25 staining to determine the DN1-DN4 fractions. Bars represent the mean ± SEM from eight mice. P-values are shown. The high side scatter cells in Hem1−/− spleens are myeloid and progenitors cells.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2585840&req=5

fig4: T cell development is severely impaired in Hem1-deficient mice. Total thymocytes (A) and splenocytes (B) from Hem1−/− and WT littermate mice were stained with fluorescent-conjugated α-CD4, α-CD8, α-CD3ε, α-CD25, α-CD44, α-B220, α-GR1, α–TCR-γδ, α–TCR-β, and intracellular α-Foxp3 and analyzed by flow cytometry. Representative histograms of eight mice per group of cells that fall within a forward scatter (FSC) and side scatter (SSC) lymphocyte gate are shown. Total thymocyte and splenocyte number were multiplied by the percentage of cells that fell within each quadrant to obtain the total number of cells within each developmental subset (right). Cells negative for B220, GR1, CD4, and CD8 were analyzed with CD44 and CD25 staining to determine the DN1-DN4 fractions. Bars represent the mean ± SEM from eight mice. P-values are shown. The high side scatter cells in Hem1−/− spleens are myeloid and progenitors cells.
Mentions: Because Hem1-deficient mice are lymphopenic, we examined whether T and/or B cell development are impaired. The development of lymphocytes can be broken down into many distinct stages based on the presence or absence of particular intracellular and surface proteins, as determined by flow cytometry (29, 30). As shown in Fig. 4 A, Hem1−/− mice have a 30-fold mean reduction in thymocyte number with an impairment in αβ T cell development within the CD4−CD8− double-negative (DN) to CD4+CD8+ double-positive (DP) cell transition at the CD25midCD44− (DN3) to CD25−CD44−(DN4) cell stage, which is mediated by formation of the preTCR. The number of γδ thymocytes is also decreased, albeit to a lesser extent than αβ T cells (Fig. 4, A and B). The total numbers of peripheral CD4, CD8, and γδ T cells (Fig. 4 B) and B lymphocytes (not depicted) are reduced by at least 75%. The ratio of Foxp3+CD4+ T regulatory cells (Treg) to Foxp3−CD4+ T cells is significantly higher in both thymus (Fig. 4 A) and spleen (Fig. 4 B), suggesting that Hem1 regulates both lymphocyte development and homeostasis.

Bottom Line: Using a chemical mutagenesis strategy in mice to identify novel genes involved in immune cell functions, we positionally cloned a nonsense mutation in the Hem1 gene.Remarkably, some Rac-dependent functions, such as Th1 differentiation and nuclear factor kappaB (NF-kappaB)-dependent transcription of proinflammatory cytokines proceed normally in Hem1-deficient mice, whereas the production of Th17 cells are enhanced.These results demonstrate that Hem1 is essential for hematopoietic cell development, function, and homeostasis by controlling a distinct pathway leading to cytoskeletal reorganization, whereas NF-kappaB-dependent transcription proceeds independently of Hem1 and F-actin polymerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
Hem1 (Hematopoietic protein 1) is a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins. Orthologues of Hem1 in Dictyostelium discoideum, Drosophila melanogaster, and Caenorhabditis elegans are essential for cytoskeletal reorganization, embryonic cell migration, and morphogenesis. However, the in vivo functions of mammalian Hem1 are not known. Using a chemical mutagenesis strategy in mice to identify novel genes involved in immune cell functions, we positionally cloned a nonsense mutation in the Hem1 gene. Hem1 deficiency results in defective F-actin polymerization and actin capping in lymphocytes and neutrophils caused by loss of the Rac-controlled actin-regulatory WAVE protein complex. T cell development is disrupted in Hem1-deficient mice at the CD4(-)CD8(-) (double negative) to CD4(+)CD8(+) (double positive) cell stages, whereas T cell activation and adhesion are impaired. Hem1-deficient neutrophils fail to migrate in response to chemotactic agents and are deficient in their ability to phagocytose bacteria. Remarkably, some Rac-dependent functions, such as Th1 differentiation and nuclear factor kappaB (NF-kappaB)-dependent transcription of proinflammatory cytokines proceed normally in Hem1-deficient mice, whereas the production of Th17 cells are enhanced. These results demonstrate that Hem1 is essential for hematopoietic cell development, function, and homeostasis by controlling a distinct pathway leading to cytoskeletal reorganization, whereas NF-kappaB-dependent transcription proceeds independently of Hem1 and F-actin polymerization.

Show MeSH
Related in: MedlinePlus