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More than 80% of BALB/c mice bearing BAMC-1 ascites tumor were completely cured after five consecutive (once every 2 days) i.p. injections of a 0.1 mg dose of OK-432, beginning on day 2 after tumor implantation. The antitumor effect of OK-432 was abolished in athymic nu/nu mice and in anti-thymocyte globulin-treated euthymic BALB/c mice, so although OK-432 treatment did increase the length of survival, all animals eventually died as a result of tumor growth. When peritoneal exudate cells (PEC), obtained on day 12 from OK-432-treated BAMC-1-bearing euthymic mice were evaluated for in vivo tumor neutralization activity, all mice receiving an i.p. injection of the admixture of the nonadherent PEC (1 X 10(7) cells) with BAMC-1 cells (1 X 10(5)) survived for more than 60 days. When the same nonadherent PEC (1 X 10(7) cells) were i.p. transferred adoptively 1 day after the inoculation of 1 X 10(5) BAMC-1 tumor cells, again all mice survived. When these in vivo active PEC were tested for cytotoxicity in vitro against fresh BAMC-1 tumor cells, natural killer (NK) sensitive syngeneic RL male 1, NK-sensitive allogeneic YAC-1 cells, NK-resistant syngeneic Meth-A cells, allogeneic tumor cells (EL4, B16, and P815) and xenogenic human cells, the PEC were found to be capable of lysing BAMC-1 tumor cells together with almost all of the other tumor cells, including NK-resistant cells. Nonadherent PEC contained at least two subpopulations of killer cells. One, directed to syngeneic BAMC-1 cells, was both Thy1.2 and asialo GM1 positive, and another, directed to allogeneic YAC-1 cells, was asialo GM1 positive but Thy1.2 negative. A cold target inhibition assay also suggested the presence of more than two subpopulations. These results indicate that T cells play a determined role in the immunotherapeutic effect of OK-432 on BALB/c mice bearing BAMC-1 tumor, although the participation of activated macrophages could not be excluded. The cells responsible for killing BAMC-1 and other tumor cells appearing in the PEC on day 12 were characterized as containing at least two kinds of lymphokine-activated killer cells.
Murine peritoneal macrophages harvested 3-4 days after IP injection of vaccinia virus lysed S91-melanoma tumor cells in vitro; enhanced tumoricidal activity was measured with effector macrophages prepared 5-6 days after vaccinia virus infection. Treatment of virus-elicited macrophages prepared from DBA/2 mice with anti-asialo-GM1 antiserum, anti-Thy 1.2 antiserum or anti-Iad antiserum in the presence of complement so that cells sensitized with antibodies were lysed, did not reduce the measured level of tumoricidal activity indicating that macrophages [Ia(-); asialo GM1(-)] and not natural killer cells [asialo GM1(+); Thy 1.2(+/-)] or T-cells [Thy 1.2(+)] were responsible for mediating the lysis of S91-melanoma tumor cells. When incubated with virus-elicited macrophages but not thioglycollate-elicited macrophages, the ability of S91-melanoma tumor cells. to synthesize DNA was completely blocked. The results of these experiments support the view that one aspect of antitumor immunity enhanced during immunotherapy with vaccinia virus is the activation of macrophages which have cytolytic as well as cytostatic effects on melanoma tumor cells.
The host cellular response to IP injection of mitomycin C was studied in C3H/HeN mice. As assessed by in vitro cytolysis assay using 125I-iododeoxyuridine-labelled tumour target cells, mitomycin C-induced peritoneal macrophages showed the maximum tumouricidal activity 4 days after the IP injection. The tumouricidal activity was dependent on the dose of mitomycin C injected and it was detectable against syngeneic, allogeneic and xenogeneic tumour target cells. In addition, these tumouricidal macrophages were found to be augmented in functions of both incorporation of 2-deoxy-D-glucose and phagocytosis of sheep red blood cells. Among the other anti-cancer drugs, which were used at a dose of three-fifths of LD50, only adriamycin (7.5 mg/kg) was capable of inducing activated macrophages as much as mitomycin C (3 mg/kg). Cyclophosphamide (225 mg/kg), methotrexate (60 mg/kg) and vincristine (1.5 mg/kg) were able to augment incorporation of 2-deoxy-D-glucose and phagocytosis of sheep red blood cells, but not tumouricidal activity. Differential cytolysis assay was performed for two cell lines of P 388 tumour target cells, the mitomycin C-sensitive original cell line and the mitomycin C-resistant subline, demonstrating no significant difference in macrophage-mediated tumour cell lysis between these cell lines. Based on these results, it was concluded that mitomycin C, when injected IP induced activated macrophages in the peritoneal cavity. A better understanding of the effect of anti-cancer drugs on macrophage tumouricidal activity may be useful in designing more effective local chemotherapy for malignant peritoneal effusions.
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Hyaluronate of 120,000 molecular weight has been injected in the peritoneal cavity of mice to study its effect on migration of inflammatory cells in vivo. After one day a dose-dependent granulocyte migration is observed. Three days later the number of granulocytes is greatly reduced and macrophages form about half of the total cell population. Hyaluronate-elicited macrophages show a decreased 5'-nucleotidase and an increased acid phosphatase activity as compared to resident macrophages. The production of superoxide anion in response to the phorbol ester tetradecanoyl-phorbolacetate, and the phagocytic activity are also enhanced. Macrophages elicited by hyaluronate secrete growth factor(s) for non-lymphoid mesenchymal cells. It is concluded that hyaluronate in vivo stimulates the migration of inflammatory cells, thus causing the recruitment of a population of stimulating macrophages. These effects may explain previous reports on the acceleration of wound healing by hyaluronate.
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