Expansion of Immunoregulatory Macrophages by Granulocyte-Macrophage Colony-stimulating Factor Derived from a Murine Mammary Tumor1

Using an immunogenic nonmetastatic murine mammary adenocarcinoma (Dl-DMBA-3) induced in BALB/c mice by dimethylbenzanthracene, we have previously shown that splenocytes from tumor bearers have depressed lymphocyte responses to mitogens and antigens, including tumor-associated antigens. In addition, they display decreased natural killer and T-cell cytotoxic activities. Macrophages from tumor-bearing mice appear to be responsible for the suppression of Tand B-cell responses to concanavalin A, lipopolysaccharide, and tumor-associated antigens observed in tumor bearers. The appearance of these macro phages in the spleen tightly parallels the progressive growth of the tumor and the concomitant immunosuppression. Simultaneously high levels of macrophage progenitors were observed in blood, bone marrow, lung, and liver. A significant increase of colony-stimulating activity of the granulocyte-macrophage lineage was detected in the sera from tumor-bearing mice. Higher levels of this colony-stimulating activity (CSA) were de tected in tumor cystic fluid as compared with the levels in serum. A tumor cell line established in vitro from the Dl-DMBA-3 in vivotumor produces high levels of a factor with CSA in culture supernatant fluids. Partial purification of the CSA from the tumor cell line supernatants was achieved using Centriceli Ultrafiltration and Sephacryl S-300 chromatography. These studies revealed that the molecular weight of the colonystimulating-like factor is 32,000 to 35,000. The morphology of the colonies obtained in cultures using this factor is similar to that of the colonies that develop in the presence of granulocyte-macrophage colonystimulating factor (GM-CSF) but not with macrophage colony-stimulat ing factor (M-CSF). CSA from tumor cell supernatants was neutralized by antiserum to GM-CSF but not with anti-M-CSF or anti-granulocyte colony-stimulating factor (G-CSF). Macrophages from bone marrow or peritoneal exÃodalesfrom normal mice cultured with tumor supernatant for 2 to 3 days strongly inhibit normal splenocyte responses to concana valin A and lipopolysaccharide. The data suggest that the tumor releases a GM-CSF that alters the hemopoietic system and induces or expands macrophages, which exert a suppressive function on the immune system of tumor-bearing mice. presser functions appear to play important roles in the regulation of a great variety of in vivo and in vitro immunological phenomena (1-7). Exactly how those cells are induced and/ or expanded in the tumor-bearing host has not been clearly elucidated. Moreover, it is not known whether tumor products and/or host products directly or indirectly cause the observed immunosuppression. Using an immunogenic nonmetastatic murine mammary adenocarcinoma induced in BALB/c mice by dimethylbenzanthracene (Dl-DMBA-3), we have previously observed a gener alized immunosuppressed state as determined by diminished proliferative responses to mitogens and antigens, decreased natural killer and cytolytic T-lymphocyte activities, and other criteria (8-11). In addition, there is an induction of macro phages in the spleen of tumor bearers which have a Mac 1*2+ phenotype. These macrophages appear to be responsible for the observed immunosuppression via various mechanisms, which include release of prostaglandins and aberrant presentation of antigens in the context of I-E molecules to T-suppressor cells (12). An investigation was undertaken to elucidate the mecha nism of induction of these suppressor macrophages that are in the spleens of mammary tumor-bearing mice. In this paper we present evidence that there is a systemic increase of granulocyte and macrophage progenitors associated with an elevated col ony-stimulating activity in the serum and tumor cystic fluid of tumor bearers. Furthermore, supernatants of tumor cells grown in vitro release high levels of GM-CSF' as defined by bioassays, biochemistry, and neutralization with antiserum to this factor. The results indicate that the tumor constitutively produces GMCSF, which induces cells of the macrophage lineage that alters nonspecific and specific immune responses in tumor-bearing