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We have investigated the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration on the leukemogenic potential of L-103 murine leukemic cells. Leukemogenic potential was assessed by comparing the regression lines drawn between the number of inoculated leukemic spleen cells and the mean survival time (MST) of the syngeneic recipients. rhG-CSF injected 2.5 μg daily for 14 days reduced the leukemogenic potential of spleen cells of the leukemic mice to 1/200 of the control. This phenomenon was not observed with the leukemic spleen cells treated with r-murine granulocyte-macrophage (rmGM)-CSF in vivo. Cytochemical study indicated that morphologically identifiable blast cells were fewer in the rhG-CSF-treated leukemic spleen. Furthermore, leukemic cells in the rhG-CSF-treated spleen were less proliferative than the control in spite of having more clonogenic cells in the leukemic cell preparation. Cytogenetical analysis showed that chromosome abnormalities found in the original leukemic cells were not altered by rhG-CSF administrations. It also showed that the frequency of the abnormal karyotype was reduced in rhG-CSF-treated leukemic spleen ( 4 17 ) as compared with the control ( 8 8 ), indicating that the mitotic fraction was smaller in the rhG-CSF-treated leukemic cells. These findings indicate that in addition to the reduced number of leukemic cells in the spleen cell preparation, a reduction of the proliferative capacity of the original leukemic cells is involved in the reduction of leukemogenic potential of leukemic cells treated with rhG-CSF in vivo.

Summary We have studied the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on granulopoiesis and neutrophil functions in aged rats and aged mice. We subcutaneously injected rG-CSF or control vehicle into aged rats (22 months old and 25 months old) for 7 consecutive days. counted the peripheral neutrophils and evaluated the functions of neutrophils isolated from venous blood. The peripheral neutrophil count in aged rats tended to be increased as compared with that in young rats (11 weeks old). However, the neutrophils in aged rats exhibited a decline of superoxide anion (O2--) release and phagocytic activity as compared with young rats. The peripheral neutrophil count in aged rats was significantly increased 5–6-fold as many as the control value by rG-CSF treatment, which was accompanied by a significant enhancement of O2-release and of phagocytic activity being restored to normal levels or better. In another series of experiments, we subcutaneously injected rG-CSF or control vehicle into aged mice (24–28 months old) or young mice (8 weeks old) for 7 consecutive days, and evaluated the functions of neutrophils isolated from peritoneal cavity. The peritoneal exudate neutrophils from the aged mice exhibited a decline of phagocytic and chemo-tactic activity as compared with the young mice. These functions in both young and aged mice were significantly enhanced by rG-CSF-treatment, and these functions in rG-CSF-treated aged mice were restored to a level higher than the level in control young mice. These findings demonstrate that rG-CSF is capable of enhancing granulopoiesis and restoring the age-related decline of neutrophil functions.

We have investigated the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on hemopoietic reconstitution after bone marrow transplantation (BMT) following lethal irradiation in mice. Mice received a daily administration of 10 micrograms/kg rG-CSF or control vehicle one through 21 days after BMT. Spleen colony-forming units (CFU-S), granulocyte-macrophage colony-forming units (CFU-GM), megakaryocyte colony-forming units (CFU-Meg), and erythroid burst-forming units (BFU-E) increased in both bone marrow and spleen of the rG-CSF-treated mice as compared with the control. This increase was evident during the administration period. In spite of the increase in the progenitor cells in bone marrow and spleen, only a recovery of neutrophils was accelerated in peripheral blood. Thus rG-CSF accelerated granulopoietic recovery in the BMT mice, with an enhanced recovery of the stem cells and the progenitors for erythrocytes and megakaryocytes. These results indicate the potential clinical usefulness of rG-CSF in the treatment of patients undergoing BMT.

Administration of human granulocyte colony-stimulating factor (hG-CSF) to mice with cyclophosphamide (CPA)-induced neutropenia for 4 consecutive days from the day after the CPA dosing (100 mg/kg) resulted in a dose-dependent increase in the peripheral blood neutrophil count 6 hours after the final hG-CSF injection. Within the hG-CSF dose range of 0.1 to 10 micrograms per mouse per day, there was a strong linear relationship (r greater than .9) between the logarithm of the dose and the peripheral blood neutrophil count in the treated mice. Using the same hG-CSF preparation, 38 experiments indicated that the regression lines are highly reproducible. Such an association never occurred with intact mice, and 100 mg/kg of CPA induced the highest response to hG- CSF. This linear relationship between the two variables allows us to determine the biologic potency of a test hG-CSF preparation relative to a reference standard using a parallel line assay, with a coefficient of precision of around .2. When assayed by this bioassay procedure, which we have termed CPA-mouse assay, natural hG-CSF and recombinant hG-CSF (produced by Chinese hamster ovary cells) were nearly equipotent in specific biologic activity. These results confirm the CPA-mouse assay as an especially useful assay method for quantifying the in vivo activity of hG-CSF.

We studied the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on leukemia development and survival of mice with leukemia by using a radiation-induced myeloid leukemia cell line (C2M) with A-type phosphoglycerate kinase (PGK) as marker isoenzyme. C3H/He mice with B-type PGK were inoculated with 2 × 106 C2M cells through the tail vein. From the next day, they received a daily subcutaneous injection of 1 μg rhG-CSF or control solution. The survival of rhG-CSF-treated recipients of C2M was significantly longer than control-solution-treated recipients. In rhG-CSF-treated recipients, not only spleen weight but also the number of blasts in hemopoietic organs was less than those in control recipients. While there was a remarkable decrease in bone marrow content of CFU-GM in control recipients, the content in rhG-CSF-treated recipients was comparable to that in normal mice. A reduced bone marrow and spleen content of leukemic colony-forming cells (L-CFU) was observed in rhG-CSF-treated recipients in comparison with control recipients. The electrophoretic analysis of PGK phenotypes of hemopoietic organs indicated the delayed appearance of A-type PGK from C2M cells in rhG-CSF-treated recipients. From these findings, we concluded that the injection of rhG-CSF delayed the onset of leukemia and improved the survival and several hematological parameters by suppressing C2M cells in recipient mice.

A colony-stimulating factor (CSF) has been purified to homogeneity from the serum-free medium conditioned by one of the human CSF-producing tumor cell lines, CHU-2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O-linked glycosides. Amino acid sequence determination of the molecule gave a single NH2-terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte-lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non-adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G-CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.

Mice were subcutaneously (sc) injected once a day for up to 15 days with a purified human native G-CSF sample at a dose of 2.5 μg/injection or with control samples with or without added endotoxin. In the G-CSF-treated mice, blood neutrophil counts began to rise as early as 2 hours after the first injection, reached a level 8 times above the preinjection level after 15 days of injections with marked elevation of all progenitor cell levels in spleen, and returned to normal within 48 hours after cessation of the injections. Such neutrophilia was observed even when endotoxin-resistant C3H HeJ mice were used, but not in control mice. It is possible that repeated G-CSF injections after administration of cyclophosphamide (CY) in mice could accelerate recovery of granulopoiesis with a rather transient rise in blood neutrophil counts.

A purified human granulocyte colony-stimulating factor (hG-CSF) was studied for its protective effect on the induction of neutropenia and enhanced susceptibility to microbial infections in mice receiving cyclophosphamide (CPA). A severe reduction in peripheral blood neutrophils was induced 4 days after injection with 200 mg of CPA per kg although the level normalized rapidly thereafter. When mice were injected subcutaneously once a day with 2.5 micrograms of hG-CSF beginning on the day after CPA injection, the reduction was prevented markedly, even 4 days later. On the other hand, in mice receiving CPA 4 days prior to infection, a weakened resistance to intraperitoneal challenge with a strain of Pseudomonas aeruginosa was induced. This weakened resistance was dose-dependently restored to normal by four daily injections with hG-CSF. A daily dose of 1.0 microgram was required for complete restoration, although hG-CSF did not directly inhibit bacterial growth in vitro. In hG-CSF-treated mice, morphologically mature neutrophils migrated rapidly into the peritoneal cavities where bacteria were inoculated, followed by a rapid elimination of bacteria from the locality as compared with controls. In addition, the same treatment with hG-CSF was able to protect significantly against systemic infections caused by Serratia marcescens, Escherichia coli, Staphylococcus aureus, and Candida albicans. These data show the possibility that prophylactic therapy with hG-CSF may augment the resistance of immunocompromised patients to infections.