ABSTRACT
KARAP/DAP12 is a transmembrane polypeptide with an intracytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). KARAP/DAP12 is associated with several activating cell surface receptors in hematopoietic cells. Here, we report that knockin mice bearing a nonfunctional KARAP/DAP12 ITAM present altered innate immune responses. Although in these mice NK cells are present and their repertoire of inhibitory MHC class I receptors is intact, the NK cell spectrum of natural cytotoxicity toward tumor cell targets is restricted. KARAP/DAP12 loss-of-function mutant mice also exhibit a dramatic accumulation of dendritic cells in muco-cutaneous epithelia, associated with an impaired hapten-specific contact sensitivity. Thus, despite its homology with CD3zeta and FcRgamma, KARAP/DAP12 plays a specific role in innate immunity, emphasizing the nonredundancy of these ITAM-bearing polypeptides in hematopoietic cells.
Subject(s)
Antigens, Ly , Cytotoxicity, Immunologic/genetics , Dendritic Cells/immunology , Killer Cells, Natural/immunology , Mice, Knockout/immunology , Receptors, Immunologic/deficiency , Receptors, Immunologic/genetics , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cell Line , Cell Movement/genetics , Cell Movement/immunology , Crosses, Genetic , Dendritic Cells/metabolism , Epithelial Cells/immunology , Gene Targeting , Immunophenotyping , Killer Cells, Natural/metabolism , Lectins, C-Type , Membrane Glycoproteins/biosynthesis , Membrane Proteins , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Molecular Sequence Data , Mucous Membrane/cytology , Mucous Membrane/immunology , Receptors, Immunologic/physiology , Receptors, NK Cell Lectin-Like , Sequence Deletion , Signal Transduction/genetics , Signal Transduction/immunology , Skin/cytology , Skin/immunologyABSTRACT
Natural killer (NK) cells are components of the innate immune system that can recognize and kill virally infected cells, tumor cells, and allogeneic cells without prior sensitization. NK cells also elaborate cytokines (e.g., interferon-gamma and tumor necrosis factor-alpha) and chemokines (e.g., macrophage inflammatory protein-1alpha) that promote the acquisition of antigen-specific immunity. NK cell differentiation is accompanied by the cell surface expression of a mucin-like glycoprotein bearing an NK cell-restricted keratan sulfate-related lactosamine carbohydrate, the PEN5 epitope. Here, we report that PEN5 is a post-translational modification of P-selectin glycoprotein ligand-1 (PSGL-1). The PEN5 epitope creates on PSGL-1 a unique binding site for L-selectin, which is independent of PSGL-1 tyrosine sulfation. On the surface of NK cells, the expression of PEN5 is coordinated with the disappearance of L-selectin and the up-regulation of Killer cell Ig-like Receptors (KIR). These results indicate that NK cell differentiation is accompanied by the acquisition of a unique carbohydrate, PEN5, that can serve as part of a combination code to deliver KIR(+) NK cells to specific tissues.
Subject(s)
Amino Sugars/chemistry , Killer Cells, Natural/metabolism , L-Selectin/metabolism , Membrane Glycoproteins/metabolism , Amino Sugars/immunology , Amino Sugars/metabolism , Antibodies, Monoclonal/immunology , Antigens, Surface/chemistry , Antigens, Surface/immunology , Antigens, Surface/metabolism , Epitopes/metabolism , Humans , Ligands , Membrane Glycoproteins/chemistry , Polysaccharides/chemistry , Polysaccharides/immunology , Polysaccharides/metabolismABSTRACT
PEN5 is a sulfated polylactosamine carbohydrate epitope first described in a subpopulation of mature natural killer cells. Here we report that it is also expressed in a developmentally regulated fashion in human and rat central nervous systems and that its protein carrier is P-selectin glycoprotein ligand-1 (PSGL-1), a ligand for selectins. In rat neural primary cultures, PEN5 is transiently and selectively expressed by oligodendrocyte precursor cells and marks the transition from proliferative to postmitotic stages. In concordance, in human central nervous system tumors, PEN5 is observed in a subset of oligodendrogliomas and in all pilocytic astrocytomas, a class of tumor of uncertain histogenesis. These data suggest that PEN5-PSGL-1 plays a role in the differentiation of oligodendrocytes and that pilocytic astrocytomas are likely to result from a dysregulation occurring in oligodendrocyte precursor cells at the crucial stage of exit from the cell cycle.
Subject(s)
Amino Sugars/biosynthesis , Antigens, Surface/biosynthesis , Astrocytoma/metabolism , Brain Neoplasms/metabolism , Oligodendroglia/metabolism , Polysaccharides/biosynthesis , Stem Cells/metabolism , Animals , Animals, Newborn , Blotting, Western , Carrier Proteins/metabolism , Cell Differentiation , Cells, Cultured , Central Nervous System/cytology , Central Nervous System/growth & development , Central Nervous System/metabolism , Embryo, Mammalian , Humans , Immunohistochemistry , Membrane Glycoproteins/metabolism , Mice , Neurons/cytology , Neurons/metabolism , Precipitin Tests , RatsABSTRACT
In humans, a minor subset of T cells express killer cell Ig-like receptors (KIRs) at their surface. In vitro data obtained with KIR(+) alphabeta and gammadelta T-cell clones showed that engagement of KIR molecules can extinguish T-cell activation signals induced via the CD3/T-cell receptor (TCR) complex. We analyzed the T-cell compartment in mice transgenic for KIR2DL3 (Tg-KIR2DL3), an inhibitory receptor for HLA-Cw3. As expected, mixed lymphocyte reaction and anti-CD3 monoclonal antibody (MoAb)-redirected cytotoxicity exerted by freshly isolated splenocytes can be inhibited by engagement of transgenic KIR2DL3 molecules. In contrast, antigen and anti-CD3 MoAb-induced cytotoxicity exerted by alloreactive cytotoxic T lymphocytes cannot be inhibited by KIR2DL3 engagement. In double transgenic mice, Tg-KIR2DL3 x Tg-HLA-Cw3, no alteration of thymic differentiation could be documented. Immunization of double transgenic mice with Hen egg white lysozime (HEL) or Pigeon Cytochrome-C (PCC) was indistinguishable from immunization of control mice, as judged by recall antigen-induced in vitro proliferation and TCR repertoire analysis. These results indicate that KIR effect on T cells varies upon cell activation stage and show unexpected complexity in the biological function of KIRs in vivo.
Subject(s)
Receptor-CD3 Complex, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , Animals , Clone Cells , Crosses, Genetic , Cytotoxicity, Immunologic , Humans , Lymphocyte Activation , Mice , Mice, Inbred CBA , Mice, Inbred Strains , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , Skin Transplantation/immunology , Spleen/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Cytotoxic/immunology , Transplantation, HomologousABSTRACT
NK and T lymphocytes share various cell surface receptors, including NK receptors for MHC class I molecules (NKR). NKR include killer cell Ig-like receptors (KIR) and lectin-like dimers which are composed of the invariant CD94 associated with a variety of NKG2 molecules. The combination of KIR and CD94/NKG2 dimers expressed on NK and T cell subsets defines a repertoire of MHC class I recognition. Engagement of NKR by cognate MHC class I molecules governs T and NK cell activation. We investigated the NKR distribution on NK and T cell subsets from uninfected and HIV-infected individuals, according to the clinical status, the absolute numbers of CD4+ T cells as well as the plasmatic viral load of the patients. We show that the KIR distribution on NK cells is not affected by HIV-1 infection, whereas the absolute numbers of T cells expressing specific KIR members (CD158b, p70) transiently increase in early stages of HIV infection. By contrast, the percentages of NK and T cells which express CD94 dimers increase in parallel with the disease. These results document a differential regulation of KIR and CD94 lectin-like dimers during the course of a chronic viral infection in humans and further suggest that both types of NKR are independently regulated.
Subject(s)
Acquired Immunodeficiency Syndrome/immunology , Antigens, CD/analysis , HIV-1 , Killer Cells, Natural/chemistry , Lectins, C-Type , Membrane Glycoproteins/analysis , Receptors, Immunologic/analysis , T-Lymphocytes/chemistry , Dimerization , Humans , NK Cell Lectin-Like Receptor Subfamily DABSTRACT
NK cell cytotoxicity is a fast and efficient mechanism of target cell lysis. Using transcription analysis, such as multiplex messenger assays, we show here that natural cytotoxicity exerted by the human NKL cell line correlates with mRNA accumulation of very early activator protein (AP)-1 transcription factor genes such as JunB, FosB and c-Fos. In addition, DNA-binding activities of Jun-Fos heterodimers were observed by electrophoretic mobility shift assays during the course of natural cytotoxicity. Interaction between immunoglobulin-like transcript-2/leukocyte Ig-like receptor 1 on NKL cells and HLA-B27 on target cells leads to an impairment of NKL natural cytotoxicity, which correlates with an absence of JunB, FosB, and c-Fos transcription, as well as an absence of their DNA-binding activity. Our studies thus indicate that, despite the rapidity of NK cell-mediated lysis, AP-1 transcription factor is activated during the early stage of NK cell cytolytic programs and that engagement of NK cell inhibitory receptors for MHC class I molecules impairs the very early activation of AP-1.
Subject(s)
Cytotoxicity, Immunologic/genetics , Killer Cells, Natural/immunology , Transcription Factor AP-1/biosynthesis , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Cell Line , Cytotoxicity, Immunologic/immunology , DNA/genetics , DNA/metabolism , Gene Expression Regulation/immunology , Genes, fos/genetics , Genes, jun/genetics , HLA-B Antigens/immunology , Humans , Killer Cells, Natural/metabolism , Lectins, C-Type , Leukocyte Immunoglobulin-like Receptor B1 , Nucleic Acid Hybridization/genetics , Nucleic Acid Hybridization/methods , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Immunologic/immunology , Transcription Factor AP-1/genetics , Up-Regulation/genetics , Up-Regulation/immunologyABSTRACT
BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1), the main inhibitor of plasminogen activators in plasma and in peritoneum, impairs plasmin formation that is essential for the repair processes of the mesothelium damaged by peritoneal dialysis fluids and peritonitis. The fibrogenetic cytokine transforming growth factor-beta (TGF-beta) displays variable effects on extracellular matrix remodeling enzymes and their inhibitors depending on tissues and cell lines. We previously found an unexpected stimulating effect of TGF-beta 1 on matrix metalloproteinase-9 in peritoneal mesothelial cells. In this study, we analyzed the effects of TGF-beta 1 on PAI-1 production and deposition in extracellular matrix. METHODS: We used primary cultured mesothelial cells and a recently established human peritoneal mesothelial cell line (HMrSV5). Cell-associated and secreted plasminogen activators and their inhibitors were detected and characterized by substrate gel zymography. PAI-1 was identified by reverse zymography and by Western blotting, and total PAI-1 was measured by ELISA. Secreted and cell-associated PA activity was measured by its ability to activate plasminogen into plasmin, that is, by the release of paranitroaniline from the plasmin synthetic substrate S-2251. PAI-1 mRNA accumulation was assessed by Northern blot. In vitro nuclear run-on assays were carried out to determine whether TGF-beta 1 had transcriptional effects on PAI-1 expression. Finally, the subcellular distribution of PAI-1 was analyzed by immunofluorescence and by immunogold silver staining. RESULTS: TGF-beta 1 increased PAI-1 antigen in the conditioned media of HMrSV5 cells, in a time- and concentration-dependent manner. This induced a dramatic decrease of free tPA in the cell medium and of membrane-bound uPA, and a parallel increase of high molecular weight PA-PAI complexes. Consequently, secreted and cell-associated plasminogen activator activities were considerably reduced. In primary cultured peritoneal mesothelial cells, TGF-beta 1 also induced PAI-1 secretion and the shift of tPA toward high molecular weight complexes. TGF-beta 1 increased PAI-1 mRNA in a time- and concentration-dependent manner. This effect was at least in part transcriptional since an approximately threefold increase in the rate of PAI-1 gene transcription was observed in nuclei sampled after a four-hour cell exposure to 5 ng/ml TGF-beta 1. Finally, TGF-beta 1 substantially increased the amount of intracellular and matrix-associated PAI-1. CONCLUSIONS: These results suggest that excessive TGF-beta 1 stimulated PAI-1 could prevent appropriate peritoneal healing by impairing the degradation of fibrin and of unorganized matrix components, and by interfering with cell migration.
Subject(s)
Epithelial Cells/metabolism , Extracellular Matrix Proteins/metabolism , Plasminogen Activator Inhibitor 1/metabolism , Transcriptional Activation/drug effects , Transforming Growth Factor beta/pharmacology , Blotting, Northern , Cell Line, Transformed/metabolism , Extracellular Matrix Proteins/analysis , Extracellular Matrix Proteins/genetics , Fibrin/metabolism , Fluorescent Antibody Technique , Gene Expression Regulation/drug effects , Humans , Peritoneum/cytology , Plasminogen Activator Inhibitor 1/analysis , Plasminogen Activator Inhibitor 1/genetics , Plasminogen Activators/genetics , Plasminogen Activators/metabolism , RNA, Messenger/analysis , Silver StainingABSTRACT
Subsets of T and natural killer (NK) lymphocytes express the CD94-NKG2A heterodimer, a receptor for major histocompatibility complex class I molecules. We show here that engagement of the CD94-NKG2A heterodimer inhibits both antigen-driven tumor necrosis factor (TNF) release and cytotoxicity on melanoma-specific human T cell clones. Similarly, CD16-mediated NK cell cytotoxicity is extinguished by cross-linking of the CD94-NKG2A heterodimer. Combining in vivo and in vitro analysis, we report that both I/VxYxxL immunoreceptor tyrosine-based inhibition motifs (ITIM) present in the NKG2A intracytoplasmic domain associate upon tyrosine phosphorylation with the protein tyrosine phosphatases SHP-1 and SHP-2, but not with the polyinositol phosphatase SHIP Determination of the dissociation constant, using surface plasmon resonance analysis, indicates that NKG2A phospho-ITIM interact directly with the SH2 domains of SHP-1 and SHP-2 with a high affinity. Engagement of the CD94-NKG2A heterodimer therefore appears as a protein-tyrosine phosphatase-based strategy that negatively regulates both antigen-induced T cell response and antibody-induced NK cell cytotoxicity. Our results suggest that this inhibitory pathway sets the threshold of T and NK cell activation.