ABSTRACT
The surface phenotype CD3-NK1.1+DX5+CD11c(int)B220+GR1- has been recently ascribed to a novel subset of mouse leukocytes termed interferon (IFN)-producing killer dendritic cells (IKDCs) that shares functions with natural killer (NK) cells and DCs. Interleukin-15 (IL-15) is critical for NK cells but its relationship with IKDC remained unexplored. An expression cassette encoding human IL-15 (hIL-15) has been transferred by hydrodynamic injection into the liver of mice, resulting in transient expression of the cytokine that is detectable during the first 48 h. hIL-15 hydrodynamic gene transfer resulted in an expansion of NK cells and IKDCs. Relative expansions of IKDCs were more dramatic in the IL-15 gene-transferred hepatic tissue than in the spleen. Adoptively transferred DX5+ cells comprising both NK cells and IKDCs proliferated in response to hydrodynamic injection of hIL-15, indicating that quantitative increases are at least in part the result of proliferation from already differentiated cells. Expansion is accompanied by enhanced cytolytic activity and increased expression of TRAIL and CD137 (4-1BB), without augmenting interferon-gamma production. The effects of a single hydrodynamic injection surpassed those of two intraperitoneal doses of the recombinant protein. The novel functional link between circulating IL-15 and IKDCs opens new possibilities to study the biology and applications of this minority cell subset.
Subject(s)
Genetic Therapy/methods , Interleukin-15/genetics , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Liver/immunology , Adoptive Transfer , Animals , Cytotoxicity, Immunologic , Dendritic Cells/immunology , Flow Cytometry , Genes, RAG-1 , Humans , Injections, Intravenous , Interleukin-15/metabolism , Lymphocyte Count , Mice , Mice, Inbred C57BL , Mice, Knockout , Recombinant Proteins , Reverse Transcriptase Polymerase Chain Reaction , Spleen/immunology , Transfection/methodsABSTRACT
OBJECTIVE: With the aim of obtaining monoclonal antibodies (mAbs) against mouse endothelial surface antigens, immunization of rats with a mouse-derived endothelial cell line (PY4.1) and subsequent hybridoma production were performed. MATERIALS AND METHODS: One of the mAbs produced by hybridoma EOL5F5 was selected for its surface binding to endothelial cell lines, and identification of the mAb-recognized antigen was performed by immunoprecipitation. Experiments were performed to analyze the effects of EOL5F5 on systemic administration to mice. RESULTS: EOL5F5-recognized antigen was a single band of 35 kDa under reducing and nonreducing conditions, features that do not match other known differentiation antigens with comparable tissue distribution. In vivo administration of purified EOL5F5 mAb to mice (n = 20) induced intense cutaneous purpura as well as severe but transient thrombocytopenia. Expression of EOL5F5-recognized antigen was detected on platelets from which it immunoprecipitated a moiety of identical electrophoretic pattern in SDS-PAGE, as the one recognized on endothelial cells. Immunohistochemically, EOL5F5-recognized antigen (p35) also was expressed on dermal capillaries, suggesting that, in addition to thrombocytopenia, damaging effects of the antibody on endothelial cells also might cause the observed purpura. CONCLUSIONS: Our results show induction of thrombocytopenic purpura in mice with an mAb against a single antigenic determinant expressed on both platelets and endothelium. EOL5F5 mAb injection sets the stage for useful experimental models that resemble immune thrombocytopenic purpura.
