ABSTRACT
NBXFO hybridoma cells produced both the membrane and secreted isoforms of macrophage colony-stimulating factor (M-CSF). Murine bone marrow cells stimulated by the secreted form of M-CSF (sM-CSF) became Mac1+, Mac2+, Mac3+, and F4/80+ macrophages that inhibited the growth of NBXFO cells, but not L1210 or P815 tumor cells. In cytotoxicity studies, M-CSF activated macrophages and freshly isolated macrophages killed NBXFO cells in the presence of polymyxin B, eliminating the possibility that contaminating lipopolysaccharide (LPS) was responsible for the delivery of the cytotoxic signal. Retroviral-mediated transfection of T9 glioma cells with the gene for the membrane isoform of M-CSF (mM-CSF), but not for the secreted isoform of M-CSF, transferred the ability of macrophages to kill these transfected T9 cells in a mM-CSF dose-dependent manner. Macrophage-mediated killing of the mM-CSF transfected clone was blocked by using a 100-fold excess of recombinant M-CSF. Catalase, superoxide dismutase, and the nitric oxide inhibitor, N-omega-nitro-arginine methyl ester (NAME), did not effect macrophage cytotoxicity against the mM-CSF transfectant T9 clones. T9 parental cells when cultured in the presence of an equal number of the mM-CSF transfectant cells were not killed, indicating specific target cell cytotoxicity by the macrophages. Electron microscopy showed that macrophages were capable of phagocytosizing mM-CSF bearing T9 tumor cells and NBXFO hybridoma cells; this suggested a possible mechanism of this cytotoxicity. This study indicates that mM-CSF provides the necessary binding and triggering molecules through which macrophages can initiate direct tumor cell cytotoxicity.
Subject(s)
Hybridomas , Macrophage Colony-Stimulating Factor/physiology , Macrophages/immunology , Neoplasm Proteins/physiology , Animals , Bone Marrow Cells , Cytotoxicity, Immunologic , Genetic Vectors/genetics , Hybridomas/metabolism , Macrophage Activation/physiology , Macrophage Colony-Stimulating Factor/genetics , Macrophage Colony-Stimulating Factor/pharmacology , Macrophages/drug effects , Male , Mice , Mice, Inbred DBA , Phagocytosis , Rats , Rats, Sprague-Dawley , Retroviridae/genetics , Spleen/cytology , TransfectionABSTRACT
The neonatal spleen:myeloma hybridoma cell, NBXFO, with immunosuppressive properties supported rodent hematopoietic colony formation. We identified this hybridoma to be an undifferentiated fibroblast that produced macrophage colony-stimulating factor (M-CSF). The bone marrow cells that grew in the presence of the NBXFO supernate were macrophages and were immunosuppressive towards lymphocytes. Neutralizing anti-M-CSF antibody partially inhibited the actions of the neonatal splenic suppressor cells. Neonatal splenocytes, but not the other parental cell line, FO, induced macrophage colony formation, possessed surface-associated M-CSF, and possessed M-CSF-specific transcripts. Therefore, we believe that the M-CSF-producing phenotype was contributed by a fibroblastic stromal cell and that these stromal cells could be responsible for the in situ generation of neonatal splenic suppressor cells.
