ABSTRACT
Eosinophils are differentiated in the bone marrow and transit through the blood circulation to home into tissues primarily under the regulation of IL-5. Because the number of eosinophils in the peripheral blood is relatively low under normal conditions, in vivo functional studies of eosinophils remain extremely difficult. Increasing their numbers in vivo might be useful for assessing eosinophil activities during parasite infections, allergic inflammation, and so on. Here, we provide a method for eosinophil expansion using IL-5 gene transfer by electroporation in vivo.
Subject(s)
Cell Culture Techniques/methods , Electroporation/methods , Eosinophils/cytology , Animals , Bone Marrow , Bone Marrow Cells/physiology , Cell Differentiation , Eosinophils/metabolism , Gene Transfer Techniques , Humans , Inflammation , Interleukin-5 , Leukocyte Count , Mice, Inbred BALB C , Mice, Inbred C57BLABSTRACT
In IL-5 transgenic mice (C3H/HeN-TgN(IL-5)-Imeg), in which 50% of peripheral blood leukocytes are eosinophils, the development of infection by Leishmania amazonensis was clearly suppressed. To determine mechanistically how this protozoan parasite is killed, we performed in vitro killing experiments. Either IL-4 or IFN-gamma effectively stimulated eosinophils to kill Leishmania amazonensis promastigotes, and most of the killing was inhibited by catalase but not by the NO inhibitor L-N5-(1-iminoethyl)-ornithine, suggesting that hydrogen peroxide is responsible for the killing of L. amazonensis by eosinophils. There was no significant degranulation of eosinophils in the culture, because eosinophil peroxidase was not detected in culture supernatants when L. amazonensis promastigotes were killed by activated eosinophils. Such resistance was also observed in BALB/c mice, which are highly susceptible to L. amazonensis. Expression plasmids for IL-4, IL-5, and IFN-gamma were transferred into muscle by electroporation in vivo starting 1 week before infection. Expression plasmid for IL-5 was most effective in slowing the development of infection among three expression plasmids. Expression plasmid for IL-4 was slightly effective and that for IFN-gamma had no effect on the progress of disease. These results suggest that IL-5 gene transfer into muscle by electroporation is useful as a supplementary protection method against L. amazonensis infection.
Subject(s)
Eosinophils/immunology , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-4/immunology , Interleukin-5/physiology , Leishmania/pathogenicity , Leishmaniasis/prevention & control , Ornithine/analogs & derivatives , Animals , Catalase/pharmacology , Cricetinae , Electroporation , Eosinophils/drug effects , Eosinophils/metabolism , Eosinophils/parasitology , Interleukin-4/genetics , Interleukin-5/genetics , Leishmania/drug effects , Leishmania/growth & development , Leishmaniasis/etiology , Leishmaniasis/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Transgenic , Ornithine/pharmacology , Superoxides/metabolismABSTRACT
We have established a culture system for the development of eosinophils from murine embryonic stem (ES) cells. After transferring ES cells from embryonic fibroblast cells onto macrophage colony-stimulating factor-deficient stromal cells, OP9, ES cells were cultured in the presence of interleukin (IL)-5 with either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF) for 20 d to obtain approximately 50% eosinophils. Electron microscopy confirmed the presence of crystallized major basic protein (MBP) in the granules of some of these cells. Neither IL-5, IL-3, GM-CSF nor eotaxin alone could induce eosinophils as efficiently as the conditions described above. Eotaxin induced eosinophil development in combination with either IL-3 or IL-5. Levels of GATA-1, Friend of GATA (FOG)-1, PU.1, CCAAT/enhancer binding protein (C/EBP)alpha, C/EBPbeta, IL-3 receptor alpha (IL-3Ralpha), GM-CSF receptor alpha (GM-CSFRalpha), and MBP mRNAs were increased in ES cells 10 d after transfer onto OP9 cells. In contrast, C/EBPepsilon, IL-5Ralpha, and eosinophil peroxidase mRNAs were induced in response to IL-3 and IL-5 after transfer onto OP9 cells. Eosinophils that developed in this system expressed Gr-1, F4/80, B220, CCR3, IL-3Ralpha, IL-5Ralpha, and DX5. Finally, eosinophils developed from ES cells produced reactive oxygen species in response to Leishmania as do peripheral blood eosinophils.
