ABSTRACT
This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N-4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA-binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.
Subject(s)
Antigens, Differentiation, T-Lymphocyte/immunology , Cell Differentiation/immunology , Gene Expression Regulation/immunology , Megakaryocytes/immunology , Animals , Antigens, Differentiation, T-Lymphocyte/genetics , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/immunology , Gene Expression Regulation/drug effects , Humans , Male , Megakaryocytes/cytology , Mice , Mice, Knockout , NF-E2 Transcription Factor, p45 Subunit/genetics , NF-E2 Transcription Factor, p45 Subunit/immunology , P-Selectin/genetics , P-Selectin/immunology , Ploidies , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/immunology , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Benign neutropenia, observed in different ethnic groups, is the most common form of neutropenia worldwide. A specific single nucleotide polymorphism, rs2814778, located at the promoter of the ACKR1 (previously termed DARC) gene, which disrupts a binding site for the GATA1 erythroid transcription factor, resulting in a ACKR1-null phenotype, was found to serve as a predictor of low white blood cell and neutrophil counts in African-Americans and Yemenite Jews. Individuals with benign neutropenia due to the ACKR1-null allele have been found to have an increased susceptibility to human immunodeficiency virus infection and, on the other hand, a protective effect against malaria. The associated protective effect may explain the spread of the ACKR1-null allele by natural selection. The reviewed relationships between ACKR1 polymorphism and various pathological states may have important clinical implications to individuals with and without benign neutropenia. Potential mechanisms for ACKR1 (previously termed DARC) modulation during neutrophil recruitment to inflammation, and chemokine bioavailability in the circulation and in local tissue are reviewed and discussed.
Subject(s)
Duffy Blood-Group System , GATA1 Transcription Factor , Neutropenia , Polymorphism, Single Nucleotide , Receptors, Cell Surface , Response Elements/immunology , Duffy Blood-Group System/genetics , Duffy Blood-Group System/immunology , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/immunology , Genetic Predisposition to Disease , HIV Infections/genetics , HIV Infections/immunology , HIV Infections/pathology , HIV-1/immunology , Humans , Malaria/genetics , Malaria/immunology , Malaria/pathology , Neutropenia/genetics , Neutropenia/immunology , Neutropenia/pathology , Neutrophils/immunology , Neutrophils/pathology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunologyABSTRACT
Megakaryocytes (MK) are the sole source of platelets in the body. They develop from lineage-committed hematopoietic stem and progenitor cells (HSPCs) via intermediate cells, which differ in morphology, size, ploidy, and surface phenotype. Development and maturation of MKs is governed by different transcription factors, including GATA-1, E26 transformation-specific transcription factor (ETS) family members, nuclear factor erythroid 2 transcription factor (NF-E2), and STAT3. During such challenges as acute inflammation, platelets are consumed in high numbers and must be replenished to secure survival of the host. This is achieved by integration of inflammatory signals into early MK development and depends on the STAT1-mediated enhanced translation of transcripts in stem cell-like megakaryocyte progenitors. Here, we review recent developments, which highlight the impact of inflammation on the development of platelets from HSPCs.
Subject(s)
Blood Platelets/immunology , Cytokines/immunology , Gene Expression Regulation, Developmental/immunology , Hematopoietic Stem Cells/immunology , Inflammation/immunology , Megakaryocytes/immunology , Animals , Blood Platelets/cytology , Cell Differentiation , Cell Lineage/immunology , Cell Proliferation , Cytokines/genetics , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/immunology , Hematopoietic Stem Cells/cytology , Humans , Inflammation/genetics , Inflammation/pathology , Megakaryocytes/cytology , Mice , NF-E2 Transcription Factor, p45 Subunit/genetics , NF-E2 Transcription Factor, p45 Subunit/immunology , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins c-ets/immunology , STAT Transcription Factors/genetics , STAT Transcription Factors/immunology , Signal TransductionABSTRACT
Dendritic cells (DCs) play a pivotal role in the regulation of the immune response. DC development and activation is finely orchestrated through transcriptional programs. GATA1 transcription factor is required for murine DC development, and data suggest that it might be involved in the fine-tuning of the life span and function of activated DCs. We generated DC-specific Gata1 knockout mice (Gata1-KODC), which presented a 20% reduction of splenic DCs, partially explained by enhanced apoptosis. RNA sequencing analysis revealed a number of deregulated genes involved in cell survival, migration, and function. DC migration toward peripheral lymph nodes was impaired in Gata1-KODC mice. Migration assays performed in vitro showed that this defect was selective for CCL21, but not CCL19. Interestingly, we show that Gata1-KODC DCs have reduced polysialic acid levels on their surface, which is a known determinant for the proper migration of DCs toward CCL21.
Subject(s)
Cell Movement/immunology , Chemokine CCL21/immunology , Dendritic Cells/immunology , GATA1 Transcription Factor/immunology , Lymph Nodes/immunology , Sialic Acids/immunology , Animals , Cell Movement/genetics , Chemokine CCL19/genetics , Chemokine CCL19/immunology , Chemokine CCL21/genetics , Dendritic Cells/cytology , GATA1 Transcription Factor/deficiency , Lymph Nodes/cytology , Mice , Mice, Knockout , Sialic Acids/geneticsABSTRACT
Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Bone Marrow Cells/drug effects , Cell Adhesion Molecules/genetics , Erythropoiesis/genetics , Fluorouracil/pharmacology , Hematopoietic Stem Cells/drug effects , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/pathology , Bone Marrow Transplantation , Cell Adhesion Molecules/deficiency , Cell Communication/drug effects , Female , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/immunology , Gene Expression Profiling , Gene Expression Regulation , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/pathology , Macrophages/drug effects , Macrophages/immunology , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Reticulocytes/drug effects , Reticulocytes/immunology , Reticulocytes/pathology , Signal TransductionABSTRACT
Pneumocystis pneumonia remains a common opportunistic infection in the diverse immunosuppressed population. One clear risk factor for susceptibility to Pneumocystis is a declining CD4(+) T cell count in the setting of HIV/AIDS or primary immunodeficiency. Non-HIV-infected individuals taking immunosuppressive drug regimens targeting T cell activation are also susceptible. Given the crucial role of CD4(+) T cells in host defense against Pneumocystis, we used RNA sequencing of whole lung early in infection in wild-type and CD4-depleted animals as an unbiased approach to examine mechanisms of fungal clearance. In wild-type mice, a strong eosinophil signature was observed at day 14 post Pneumocystis challenge, and eosinophils were increased in the bronchoalveolar lavage fluid of wild-type mice. Furthermore, eosinophilopoiesis-deficient Gata1(tm6Sho)/J mice were more susceptible to Pneumocystis infection when compared with BALB/c controls, and bone marrow-derived eosinophils had in vitro Pneumocystis killing activity. To drive eosinophilia in vivo, Rag1(-/-) mice were treated with a plasmid expressing IL-5 (pIL5) or an empty plasmid control via hydrodynamic injection. The pIL5-treated mice had increased serum IL-5 and eosinophilia in the lung, as well as reduced Pneumocystis burden, compared with mice treated with control plasmid. In addition, pIL5 treatment could induce eosinophilia and reduce Pneumocystis burden in CD4-depleted C57BL/6 and BALB/c mice, but not eosinophilopoiesis-deficient Gata1(tm6Sho)/J mice. Taken together, these results demonstrate that an early role of CD4(+) T cells is to recruit eosinophils to the lung and that eosinophils are a novel candidate for future therapeutic development in the treatment of Pneumocystis pneumonia in the immunosuppressed population.
Subject(s)
Eosinophils/immunology , Interleukin-5/immunology , Lung/immunology , Pneumocystis/immunology , Pneumonia, Pneumocystis/immunology , Animals , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/microbiology , CD4-Positive T-Lymphocytes/pathology , Eosinophils/microbiology , Eosinophils/pathology , Female , GATA1 Transcription Factor/deficiency , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/immunology , Gene Expression , Genetic Therapy , Homeodomain Proteins/genetics , Homeodomain Proteins/immunology , Host-Pathogen Interactions , Interleukin-5/genetics , Leukocyte Count , Lung/microbiology , Lung/pathology , Lymphocyte Activation , Lymphocyte Depletion , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Plasmids/administration & dosage , Plasmids/immunology , Pneumonia, Pneumocystis/genetics , Pneumonia, Pneumocystis/pathology , Pneumonia, Pneumocystis/therapy , Time FactorsABSTRACT
Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by neutrophil dysfunction, bilobed neutrophil nuclei and lack of neutrophil-specific granules. Defects in a myeloid-specific transcription factor, CCAAT/enhancer binding protein-ε (C/EBPε), have been identified in two cases in which homozygous frameshift mutations led to loss of the leucine zipper domain. In this study, we report a 55-y-old woman affected with SGD caused by a novel homozygous 2-aa deletion (ΔRS) in the leucine zipper domain of the C/EBPε gene. The patient showed characteristic neutrophil abnormalities and recurrent skin infections; however, there was no history of deep organ infections. Biochemical analysis revealed that, in contrast to the two frameshift mutations, the ΔRS mutant maintained normal cellular localization, DNA-binding activity, and dimerization, and all three mutants exhibited marked reduction in transcriptional activity. The ΔRS mutant was defective in its association with Gata1 and PU.1, as well as aberrant cooperative transcriptional activation of eosinophil major basic protein. Thus, the ΔRS likely impairs protein-protein interaction with other transcription factors, resulting in a loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function, and indicate that multiple molecular mechanisms lead to SGD.
Subject(s)
Base Sequence , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/immunology , Lactoferrin/deficiency , Leukocyte Disorders/genetics , Neutrophils/immunology , Sequence Deletion , Adult , Cytoplasmic Granules/immunology , Cytoplasmic Granules/pathology , Eosinophil Major Basic Protein/genetics , Eosinophil Major Basic Protein/immunology , Female , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/immunology , Gene Expression Regulation , Homozygote , Humans , Lactoferrin/genetics , Lactoferrin/immunology , Leukocyte Disorders/immunology , Leukocyte Disorders/pathology , Male , Middle Aged , Molecular Sequence Data , Neutrophils/pathology , Protein Binding , Protein Structure, Tertiary , Proteoglycans/genetics , Proteoglycans/immunology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/immunology , Signal Transduction , Trans-Activators/genetics , Trans-Activators/immunology , Transcription, GeneticABSTRACT
Human peripheral blood natural killer progenitors represent a flexible, heterogeneous population whose phenotype and function are controlled by their membrane-bound IL-15. Indeed, reciprocal membrane-bond IL-15 trans-presentation commits these cells into NK differentiation, while membrane-bound IL-15 stimulation with its soluble ligand (sIL-15Rα) triggers a reverse signal (pERK1/2 and pFAK) that modifies the developmental program of at least two subsets of PB-NKPs. This treatment generates: i) the expansion of an immature NK subset growing in suspension; ii) the appearance of an unprecedented adherent non-proliferative subset with a dendritic morphology co-expressing marker, cytokines and functions typical of myeloid dendritic cells (CD1a(+)/BDCA1(+)/IL-12(+)) and NK cells (CD3-/NKp46(+)/ CD56(+)/IFNγ(+)). The generation of these putative NK/DCs is associated to the rapid inhibition of negative regulators of myelopoiesis (the transcription factors STAT6 and GATA-3) followed by the transient upregulation of inducers of myeloid development, such as the transcription factors (PU.1, GATA-1) and the anti-apoptotic molecule (MCL-1).
Subject(s)
Dendritic Cells/immunology , Hematopoietic Stem Cells/immunology , Interleukin-15/immunology , Killer Cells, Natural/immunology , Membrane Proteins/immunology , Blotting, Western , Cell Adhesion/immunology , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cells, Cultured , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/metabolism , Flow Cytometry , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/immunology , GATA1 Transcription Factor/metabolism , GATA3 Transcription Factor/immunology , GATA3 Transcription Factor/metabolism , Hematopoietic Stem Cells/metabolism , Humans , Interleukin-15/metabolism , Killer Cells, Natural/metabolism , Lymphocyte Culture Test, Mixed , Membrane Proteins/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Myeloid Cells/immunology , Myeloid Cells/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/immunology , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Interleukin-15/immunology , Receptors, Interleukin-15/metabolism , Reverse Transcriptase Polymerase Chain Reaction , STAT6 Transcription Factor/immunology , STAT6 Transcription Factor/metabolism , Signal Transduction/drug effects , Signal Transduction/immunology , Stem Cell Factor/pharmacologyABSTRACT
Eosinophil/basophil (Eo/B) progenitor phenotype and function in cord blood (CB) are associated with atopic risk at birth and infant clinical outcomes. Molecular analyses of eosinophil-basophil differentiation events could identify clinically predictive biomarkers. To determine CB kinetic patterns of Eo/B lineage-associated gene expression (GATA-1, MBP1 and IL-5R alpha) after IL-5 stimulation, CB non-adherent mononuclear cells were isolated from random fresh and frozen samples and incubated in the presence of recombinant human interleukin-5. Some underwent CD34+ positive selection using magnetic cell separation. At various time-points, mRNA expression of GATA-1, MBP1 and IL-5R alpha (total transcripts) was determined utilizing multiplex quantitative polymerase chain reaction (Q-PCR). Relative expression levels of the IL-5R alpha soluble vs. transmembrane isoforms were also analyzed. Stimulation of the non-adherent mononuclear cells with IL-5 resulted in early up-regulation of GATA-1, peaking at 48 h, followed by decreasing expression and down-regulation by 96 h. The CD34+ enriched population demonstrated an equivalent expression pattern (r = 0.963, p = 0.0349). MBP1 mRNA expression [non-adherent mononuclear cells (NAMNCs) and CD34+ alike; r = 0.988, p = 0.012] was slowly up-regulated in response to IL-5, maximal at 96 h. Total IL-5R alpha expression appeared stable over the time-course, mediated by differential expression of the soluble and transmembrane isoforms (i.e., initial increase in the transmembrane contribution followed by a predominance of the soluble isoform by 48-72 h). Multiplex Q-PCR analysis of mRNA from CB demonstrates expression of critical eosinophil-basophil lineage-specific events that are consistent with current understanding of eosinophil differentiation and maturation. The non-adherent mononuclear cell population provides a surrogate signal for the CD34+ progenitor population.
Subject(s)
Biomarkers/metabolism , DNA-Binding Proteins/metabolism , Eosinophils/metabolism , GATA1 Transcription Factor/metabolism , Interleukin-5 Receptor alpha Subunit/metabolism , Antigens, CD34/biosynthesis , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Eosinophils/cytology , Eosinophils/immunology , Fetal Blood/cytology , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/immunology , Gene Expression Regulation/immunology , Hematopoiesis/genetics , Hematopoiesis/immunology , Humans , Infant, Newborn , Interleukin-5/immunology , Interleukin-5/metabolism , Interleukin-5 Receptor alpha Subunit/genetics , Interleukin-5 Receptor alpha Subunit/immunology , RNA, Messenger/analysis , Time FactorsABSTRACT
We describe a patient, who developed during the first week of life petechiae and hematomas caused by severe thrombocytopenia and gastrointestinal bleeding due to multiple small gastric hemangiomata. Bone marrow examination showed hypermegakaryocytosis and dysmegakaryopoiesis. Alloimmune thrombocytopenia was excluded. Only 3 y later, platelet counts normalized and bleedings disappeared but small skin hemangiomata remained. Electron microscopy showed enlarged round platelets with a paucity of alpha granules similar as in GATA1-deficient patients but no GATA1 mutation was found. Immunoblot analysis showed a strong interaction between patient Igs and recombinant GATA1, GATA2, and the N finger (Nf) of GATA1. The lymphocyte transformation test with recombinant GATA1Nf was positive. In vitro culturing of normal CD34 cells with purified patient Igs showed a decreased number of megakaryocyte colonies but an increased overall size of the colonies compared with control Igs. Mice injected with patient Igs showed a reduced platelet count compared with mice injected with control Igs. Thrombopoiesis was also reduced after injection of patient Igs in transgenic zebrafish compared with control Igs. In conclusion, this study is the first report of an anti-GATA1 autoantibody leading to severe thrombocytopenia and gastrointestinal bleeding from multiple pinpoint hemangiomata.
Subject(s)
Autoantibodies/blood , Autoimmunity , GATA1 Transcription Factor/immunology , Gastrointestinal Hemorrhage/immunology , Gastrointestinal Neoplasms/immunology , Hemangioma/immunology , Thrombocytopenia/immunology , Animals , Animals, Genetically Modified , Cells, Cultured , Child, Preschool , Female , GATA1 Transcription Factor/genetics , Gastrointestinal Hemorrhage/blood , Gastrointestinal Hemorrhage/therapy , Gastrointestinal Neoplasms/blood , Gastrointestinal Neoplasms/therapy , Hemangioma/blood , Humans , Infant, Newborn , Lymphocytes/immunology , Mice , Mice, Inbred NOD , Mice, SCID , Platelet Count , Severity of Illness Index , Thrombocytopenia/blood , Thrombocytopenia/therapy , Thrombopoiesis , Transfection , Zebrafish/geneticsABSTRACT
Two major pathways of human myeloid dendritic cell (DC) subset differentiation have previously been delineated. Langerhans cells (LCs) reside in epithelia in the steady state, whereas monocytes can provide dendritic cells (DCs) on demand in response to inflammatory signals. Both DC subset pathways arise from shared CD14+ monocyte precursors, which in turn develop from myeloid committed progenitor cells. However, the underlying hematopoietic mechanisms still remain poorly defined. Here, we demonstrate that the vitamin D(3) receptor (VDR) is induced by transforming growth factor beta1 during LC lineage commitment and exerts a positive role during LC generation. In contrast, VDR is repressed during interleukin-4 (IL-4)-dependent monocyte-derived DC (moDC) differentiation. We identified GATA-1 as a repressor of VDR. GATA-1 is induced by IL-4 in moDCs. Forced inducible expression of GATA-1 mimics IL-4 in redirecting moDC differentiation and vice versa, GATA-1 knockdown arrests moDC differentiation at the monocyte stage. Moreover, ectopic GATA-1 expression stabilizes the moDC phenotype under monocyte-promoting conditions in the presence of vitamin D3 (VD3). In summary, human myeloid DC subset differentiation is inversely regulated by GATA-1 and VDR. GATA-1 mediates the repression of VDR and enables IL-4-dependent moDC differentiation. Conversely, VDR is induced downstream of transforming growth factor beta1 and is functionally involved in promoting LC differentiation.
Subject(s)
Cell Differentiation/immunology , Dendritic Cells/immunology , GATA1 Transcription Factor/immunology , Monocytes/immunology , Myeloid Progenitor Cells/immunology , Receptors, Calcitriol/immunology , Repressor Proteins/immunology , Cell Differentiation/drug effects , Dendritic Cells/cytology , Dendritic Cells/metabolism , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/metabolism , Gene Knockdown Techniques , Humans , Interleukin-4/genetics , Interleukin-4/immunology , Interleukin-4/pharmacology , K562 Cells , Lipopolysaccharide Receptors , Monocytes/cytology , Monocytes/metabolism , Myeloid Progenitor Cells/cytology , Myeloid Progenitor Cells/metabolism , Receptors, Calcitriol/genetics , Receptors, Calcitriol/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/immunology , Transforming Growth Factor beta1/pharmacology , U937 CellsABSTRACT
Currently, the origin of autoimmune diseases is considered to be multifactorial. Genetic predisposition, immune system malfunction or even backfire, hormonal regulation, and environmental factors, i.e. infections, all play important roles in the pathogenesis of autoimmune diseases such as the antiphospholipid syndrome (APS). New drugs and strategies aimed at preventing infections could further improve the outcome of APS and other autoimmune diseases.
Subject(s)
Antibodies, Antiphospholipid/immunology , Antiphospholipid Syndrome/etiology , DNA Virus Infections/complications , DNA Viruses/immunology , Gram-Positive Bacteria/immunology , Gram-Positive Bacterial Infections/complications , Antibodies, Antineutrophil Cytoplasmic/immunology , Antigens, Bacterial/immunology , Antigens, Viral/immunology , Antiphospholipid Syndrome/physiopathology , Autoantigens/immunology , Autoimmunity , DNA Virus Infections/immunology , DNA Viruses/pathogenicity , GATA1 Transcription Factor/immunology , Gram-Positive Bacteria/pathogenicity , Gram-Positive Bacterial Infections/immunology , Humans , Immunity, Innate , Molecular MimicryABSTRACT
D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.
Subject(s)
GATA1 Transcription Factor/immunology , Gene Expression Regulation/immunology , Hematopoietic Stem Cells/immunology , Leukocytes/immunology , Models, Biological , Receptors, CCR10/immunology , Animals , Chemokines/genetics , Chemokines/immunology , Chemokines/metabolism , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/metabolism , Gene Expression Regulation/genetics , Hematopoietic Stem Cells/cytology , Humans , Leukocytes/metabolism , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Mice , Organ Specificity/genetics , Organ Specificity/immunology , Receptors, CCR10/biosynthesis , Receptors, CCR10/genetics , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/pharmacology , Chemokine Receptor D6ABSTRACT
Cell-type-specific transcription of mouse high-affinity IgE receptor (FcepsilonRI) beta-chain is positively regulated by the transcription factor GATA-1. Although GATA-1 is expressed in erythroid cells, megakaryocytes, and mast cells, the expression of mouse FcepsilonRI beta-chain is restricted to mast cells. In the present study, we characterized the role of GATA-associated cofactor FOG-1 in the regulation of the FcepsilonRI beta-chain promoter. The expression levels of FOG-1, GATA-1, and beta-chain in each hematopoietic cell line were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. FOG-1 expression was higher in the beta-chain-negative hematopoietic progenitor cell line Ba/F3 than in the beta-chain-positive mast cell line PT18. By contrast, GATA-1 expression was similar when comparing the 2 cell lines. A transient reporter assay demonstrated that the beta-chain promoter functioned in PT18 but not in Ba/F3 and that the transcription activity of the beta-chain promoter in PT18 was markedly suppressed by overexpression of FOG-1. Although the activity of the beta-chain promoter, which was upregulated by coexpression of GATA-1, was significantly suppressed by coexpression of FOG-1 in the simian kidney CV-1 cells (beta-chain(-), GATA-1(-), and FOG-1(-)), the transactivation of the beta-chain promoter by the GATA-1 mutant V205G, which cannot bind FOG-1, was not affected by coexpression of FOG-1. Further, overexpression of FOG-1 in PT18 resulted in decreases in cell surface expression of FcepsilonRI and beta-chain transcription. Finally, suppression of FOG-1 expression using an siRNA approach resulted in increased beta-chain promoter activity in Ba/F3. These results suggest that FOG-1 expression level regulates the GATA-1-dependent FcepsilonRI beta-chain promoter.
Subject(s)
GATA1 Transcription Factor/antagonists & inhibitors , Gene Expression Regulation/genetics , Mast Cells/immunology , Nuclear Proteins/pharmacology , Receptors, IgE/antagonists & inhibitors , Receptors, IgE/genetics , Transcription Factors/pharmacology , Transcription, Genetic , Animals , Cell Line , Down-Regulation/drug effects , Down-Regulation/immunology , GATA1 Transcription Factor/immunology , Gene Expression Regulation/drug effects , Mice , Nuclear Proteins/biosynthesis , Nuclear Proteins/immunology , Receptors, IgE/immunology , Structure-Activity Relationship , Transcription Factors/biosynthesis , Transcription Factors/immunologyABSTRACT
CC chemokine receptor 5 (CCR5) is the major HIV-1 coreceptor and its expression levels are a critical determinant of HIV-1 infection. However, the molecular mechanisms of CCR5 regulation in primary targets of HIV-1 remain unknown. Despite binding to conserved DNA elements, we show that the transcription factors GATA binding protein 1 (GATA-1) and GATA-3 differentially suppress the expression of CCR5 in stem-cell-derived dendritic cells and primary human T-cell subsets. In addition, GATA-1 expression was also more potent than GATA-3 in suppressing T helper 1 (Th1)-associated genes, interferon-gamma (IFNgamma), and CXC chemokine receptor-3 (CXCR3). GATA-1, but not GATA-3, potently suppressed CCR5 transcription, thereby rendering human T cells resistant to CCR5-tropic HIV-1 infection. However, GATA-1 could also serve as a surrogate for GATA-3 in its canonic role of programming Th2 gene expression. These findings provide insight into GATA-3-mediated gene regulation during T-cell differentiation. Importantly, decoding the mechanisms of GATA-1-mediated repression of CCR5 may offer an opportunity to develop novel approaches to inhibit CCR5 expression in T cells.