Takuji Kawashima

Augmentation of Cancer Chemotherapy by Preinjection of Human Macrophage Colony‐stimulating Factor in L1210 Leukemic Cell‐inoculated Mice

Human macrophage colony‐stimulating factor (hM‐CSF) is a potent stimulator of the effector functions of monocytes/macrophages. We investigated the antitumor effects of this factor in CDF1 male mice inoculated with L1210 cells, a mouse B‐cell leukemia line. Mice preinoculated with various numbers of L1210 cells on day 0 were given intravenous injections of vehicle (human serum albumin; HSA) (100μg/kg/day) or hM‐CSF (20μg/kg/day) for 3 days from day 1. In mice preinoculated with 102 L1210 cells but not with 103 or more L1210 cells, a marked increment in survival rate was observed with hM‐CSF treatment. We next examined the effect of hM‐CSF treatment combined with chemotherapy on the survival of mice that had been preinoculated with 105 L1210 cells. In our system, the administration of 4.9 mg/kg adriamycin (ADM) alone slightly prolonged survival of the tumorbearing mice, but all of the mice died within 20 days. When hM‐CSF was injected for 3 days before this ADM treatment, the invasion and proliferation of tumor cells in the liver and spleen were markedly inhibited and 50% of the mice were still alive at day 50. We detected inhibitory activity toward L1210 growth in serum of mice administered with hM‐CSF, and the degree of the inhibitory activity was correlated with the level of nitrite (NO2) in the serum. When L1210 cells were co‐cultured with peritoneal macrophages from mice intraperitoneally injected with hM‐CSF, the uptake of [3H]thymidine in L1210 cells was inhibited. The inhibition was abolished by the addition of NG‐monomethyI‐L‐arginine, an inhibitor of NO2− synthesis, suggesting that the reactive nitrogen oxide intermediate is involved in hM‐CSF‐induced inhibition of L1210 growth.

Effect of Human Macrophage Colony‐stimulating Factor on Granulopoiesis and Survival in Bone‐marrow‐transplanted Mice

Human macrophage colony‐stimulating factor (hM‐CSF) has been isolated from normal human urine and purified to a homogenous protein. The effect of hM‐CSF on granulopoiesis was investigated in BALB/c mice transplanted with a suboptimal number of bone marrow cells. Lethally irradiated (7.8 Gy) mice were transplanted with 1 × 106 syngeneic mouse bone marrow cells and treated with a daily intraperitoneal dose of 64 μg/kg of hM‐CSF for 5 days following the transplant. The hM‐CSF injection resulted in stimulation of the recovery of blood neutrophils as well as an increase in the number of granulocyte‐macrophage progenitor cells (CFU‐GM) in the femur and spleen. The survival of lethally irradiated mice was dependent on the cell number transplanted; most mice transplanted with 2 × 104 cells died within 2 weeks. The recovery of hematopoiesis in mice transplanted with 2 × 104 cells was modestly but significantly stimulated by hM‐CSF administration initiated from 5 days before or 1 day after transplantation for a 5‐day period. Furthermore, the hM‐CSF administrations markedly reduced the mortality in these mice during the early period after the transplantation. Since anaerobic bacteria were frequently detected in arterial blood immediately before the deaths but were not found in the surviving mice, it is speculated that early deaths occurring within 2 weeks after the transplant may be caused by opportunistic infections, and hM‐CSF injection may prevent these mortal infections through its stimulating effect on monocyte‐macrophage functions that are responsible for the production of hematopoietic regulators.

Proteoglycan form of macrophage colony-stimulating factor binds low density lipoprotein.

We recently isolated a proteoglycan form of macrophage colony-stimulating factor (PG-M-CSF) that carries a chondroitin sulfate glycosaminoglycan chain. Here, we examined the interaction of PG-M-CSF with low density lipoprotein (LDL). When LDL preincubated with PG-M-CSF was fractionated by molecular size sieving chromatography, it was eluted earlier than untreated LDL. When LDL was preincubated with chondroitin sulfate-free 85-kD M-CSF instead of PG-M-CSF, the elution profile of LDL remained unchanged, indicating specific interaction between PG-M-CSF and LDL. The level of PG-M-CSF binding in the wells of a plastic microtitration plate precoated with LDL was significant, this binding being completely abolished by pretreatment of PG-M-CSF with chondroitinase AC, which degrades chondroitin sulfate. The addition of exogenous chondroitin sulfate or apolipoprotein B inhibited the binding of PG-M-CSF to LDL in a dose-dependent manner, indicating that the interaction between PG-M-CSF and LDL was mediated by the binding of the chondroitin sulfate chain of PG-M-CSF to LDL apolipoprotein B. PG-M-CSF was also demonstrated in the arterial wall, and there were increased amounts of PG-M-CSF in atherosclerotic lesions. The in vitro interaction between PG-M-CSF and LDL thus appears to have physiological significance.