Mature and immature myeloid cells decrease the granulocyte colony-stimulating factor level by absorption of granulocyte colony-stimulating factor.

We studied the effects of polymorphonuclear neutrophils (PMN) and immature myeloid cells on the granulocyte colony-stimulating factor (G-CSF) level in vitro to better understand the regulatory mechanisms of neutropoiesis. Intact normal PMN decreased the G-CSF level after incubation with recombinant human (rh) G-CSF in a time- and dose-dependent manner. The percent reduction decreased as the concentration of rhG-CSF increased. However, the cell-free PMN-conditioned medium (PMN-CM) did not decrease the G-CSF level. The intact PMN also decreased the granulocyte-macrophage (GM)-CSF level after culture with rhGM-CSF, but did not affect the monocyte (M)-CSF level after culture with rhM-CSF. Normal bone marrow (BM) immature neutrophilic cells and G-CSF-dependent acute myeloid leukemic cells (OCI/AML la) also decreased the G-CSF level, whereas K-562 cells, which have no detectable G-CSF receptors, did not affect it. Phenylarsine oxide (PhAsO), an inhibitor of endocytosis of ligand receptor complex, abrogated this decreasing effect of intact PMN and OCI/AML la cells. These findings suggest that mature and immature myeloid cells negatively regulate neutropoiesis by, at least in part, decreasing the G-CSF level probably through receptor-mediated continual absorption and metabolism of G-CSF.

Exposure-based safety evaluation of recombinant human macrophage colony-stimulating factor (M-CSF) in cynomolgus monkeys.

The safety of M-CSF was assessed in cynomolgus monkeys in an intravenous dosing regimen. Exposure (AUC0-24) multiples (monkey vs human) were calculated using the no observable adverse effect level (NOAEL) observed in this study and correlated with known M-CSF-induced toxicities in a previous continuous intravenous infusion (civ) study in monkeys. M-CSF was administered by daily intravenous infusion (2 h) to cynomolgus monkeys (2/sex/ group) at 0.1, 0.3, 0.7, and 1.0 mg/kg/day, for 28 consecutive days. Control animals (2/sex) received placebo. The 0.7 mg/kg/day group was held for an additional 4-week recovery period. Criteria evaluated included physical observations, ophthalmoscopy, electrocardiography, body weight, food consumption, clinical pathology, antibody formation, pharmacokinetics, necropsy, organ weights, and histopathology. The only effect previously seen in monkeys after intravenously administered M-CSF occurred in animals in the 0.7 and 1.0 mg/kg/day groups. They exhibited a slight decrease in platelets between days 4 and 12 with subsequent recovery. No effects related to M-CSF administration were evident in macroscopic or microscopic evaluations and there was no evidence of anti-M-CSF antibody production. M-CSF at all dose levels was completely eliminated within each dosing interval with no accumulation. Clearance of M-CSF was enhanced during the first week of dosing, but returned to baseline clearance levels by day 27. This dosing regimen was shown to be remarkably free of toxicities noted in a previous monkey study where M-CSF was given by civ at similar daily doses. At the high dose, which was considered to be the NOAEL, the AUC0-24 was 40-fold greater than the AUC0-24 in clinical trials where 2.0 mg/m2 was administered by a daily 2-h infusion.

The effects of colony-stimulating factor-1 on the distribution of mononuclear phagocytes in the developing osteopetrotic mouse.

Colony-stimulating factor-1 (CSF-1), the primary regulator of mononuclear phagocyte (Mphi) production, exists as either a circulating or cell surface, membrane-spanning molecule. To establish transplacental transfer of maternal CSF-1, gestational day-17 mothers were injected intravenously with 125I-mouse CSF-1 or human rCSF-1, and the 125I-cpm or human CSF-1 concentrations were measured in fetal tissue, placenta, and fetal/maternal sera. Biologically active CSF-1 crossed the placenta and peaked in fetal tissue, placenta, and serum 10 minutes after injection. The role of CSF-1 in perinatal Mphi development was examined by studying the CSF-1-deficient osteopetrotic (csfmop/csfmop) mouse. Fetal/neonatal mice, derived from matings of either +/csfmop females with csfmop/csfmop males or the reciprocal pairings, were genotyped and tissue Mphi identified and quantified. In the presence of circulating maternal CSF-1 (+/csfmop mother), Mphi development in csfmop/csfmop liver was essentially complete at birth relative to +/csfmop littermates, but significantly reduced in spleen, kidney, and lung. In the absence of circulating maternal CSF-1 (csfmop/csfmop mother), Mphi numbers at birth were reduced in csfmop/csfmop liver relative to the offspring of +/csfmop mothers, but were similar in spleen, kidney, and lung. We conclude that CSF-1 is required for the perinatal development of most Mphi in these tissues. Compensation for total absence of local CSF-1 production by circulating, maternal CSF-1 is tissue-specific and most prominent in liver, the first fetal organ perfused by placental blood. However, because some Mphi developed in the complete absence of CSF-1, other factors must also be involved in the regulation of macrophage development.

In vitro expansion of murine multipotential hematopoietic progenitors from the embryonic aorta-gonad-mesonephros region.

The origin of hematopoietic stem cells (HSCs) and their growth factor requirement are poorly understood. Here we describe a new in vitro culture system of the aorta-gonad-mesonephros (AGM) region, where long-term repopulating HSCs first arise. We demonstrate that oncostatin M (OSM) is expressed in the AGM and is absolutely required for the expansion of multipotential hematopoietic progenitors in vitro. In addition, OSM enhances the formation of endothelial cell clusters. Thus, OSM appears to be a key cytokine for the development of multipotential hematopoietic progenitors in the AGM, possibly acting on common precursor cells between HSCs and endothelial cells. By using the AGM culture derived from macrophage colony-stimulating factor (M-CSF)-deficient op/op mutant embryos, we also show a pivotal role for M-CSF in fetal myelopoiesis.

Intravenously administered macrophage colony-stimulating factor (M-CSF) specifically acts on the spleen, resulting in the increasing and activating spleen macrophages for cytokine production in mice.

IL-6 was transiently expressed in sera of mice after a bolus intravenous injection with LPS and it peaked 2 h later. Intravenous administration of M-CSF at 250 micrograms/kg/day for 5 days prior to an injection of 25 micrograms/kg of LPS elevated the serum IL-6 level 10-fold higher than that of mice which were not given M-CSF. Although M-CSF had no effect on the number of macrophages in alveoli and peritoneal cavity, it tripled the number of spleen macrophages and increased macrophage-progenitor cells 7-fold when injected intravenously. Spleen macrophages from M-CSF-injected mice produced 5-fold more IL-6 in response to LPS-stimulation in-vitro. However, M-CSF-injection had lesser effects on LPS-induced IL-6 production from liver, alveolar and peritoneal macrophages. Exogenously administered M-CSF was detected at higher concentration and for longer duration in the spleen than in any other organs examined. Spleen macrophages incubated in-vitro with more than 1000 U/ml of M-CSF for 3 days also produced more LPS-induced IL-6 than untreated cells. These results indicate that intravenously administered M-CSF not only enhances macrophage development in the spleen, but also primes mature macrophages for cytokine production.

Structure-function studies on human macrophage colony-stimulating factor (M-CSF).

M-CSF (CSF-1) can be produced in a variety of structural forms that may affect function in vivo. Truncated, nonglycosylated forms of recombinant M-CSF (rM-CSF) from E. coli have been refolded in vitro in high yield and shown to be functionally equivalent in vitro to glycosylated rM-CSF secreted from mammalian cells. An N-terminal domain of 149 amino acids is produced by all of the known M-CSF mRNA splice variants and is the region responsible for bioactivity observed in vitro. Heterodimeric rM-CSFs from different splice variants containing this domain were produced in pure form by refolding in vitro, and are fully active, but have yet to be observed in vivo. The circulating half-life of truncated M-CSF forms injected intravenously into rats increased with the MW of the M-CSF used. Large increases in half-life in vivo were observed following chemical addition of a single molecule of 10 kD polyethylene glycol to rM-CSF in vitro. The crystal structure of rM-CSF revealed that M-CSF is a member of a family of molecules related by having a distinctive four-helical-bundle structural core. Site-directed mutagenesis showed that residues in or near helix A and helix C are involved in receptor binding, as reflected by decreased bioactivity and receptor binding of certain mutants. A soluble form of the M-CSF receptor, c-fms, was produced in a baculovirus/Sf9 expression system and purified to homogeneity. The MW of rM-CSF saturated with this soluble receptor was determined by molecular sieve chromatography and light scattering. Each dimeric M-CSF molecule appears to bind two soluble receptor molecules in vitro, supporting the observation that M-CSF signaling is linked to receptor dimerization.

Kinetics and dose dependence of macrophage colony-stimulating factor-induced proliferation and activation of murine mononuclear phagocytes in situ: differences between lungs, liver, and spleen.

Alveolar macrophages (AM) play an important role in antimicrobial defense mechanisms of the lung. It therefore seems reasonable to use macrophage colony-stimulating factor (M-CSF) to enhance local resistance mechanisms. However, little is known about the in vivo activity of M-CSF on macrophages in various organs. We determined the effect of a single subcutaneous dose of M-CSF (10, 50, 100, and 500 ng, respectively) on the number and functional status of AM as well as of macrophages in liver and spleen of mice. Organs were investigated immunohistochemically on days 1 and 3 after injection using monoclonal antibodies specific for F4/80, Ia antigen, and MAC-1. We found a significant increase in the number of F4/80+ AM, Kupffer cells, and splenic macrophages reaching its maximum 24 h after injection of low doses (10 and 50 ng per mouse, respectively) of M-CSF and decreasing to a level seen in untreated mice at 72 h after M-CSF in liver and spleen, whereas at a dose of 50 ng per mouse the number of AM remained high. In contrast, the numbers of AM, Kupffer cells, and splenic macrophages did not increase significantly when high doses were used (500 ng). The expression of Ia antigen and MAC-1 was increased on macrophages in the spleen but not on AM or Kupffer cells. TNF-alpha was elevated in bronchoalveolar (BAL) fluid after 3 h and IL-6 at 6, 12, and 24 h after M-CSF injection in dose-dependent manner. Nitric oxide production was not increased after injection of M-CSF. Our results point to regional differences in the response of macrophages to M-CSF. These may caused by differences in the M-CSF-induced production of TNF-alpha and IL-6. These findings may be important for the therapeutic use of M-CSF in microbial infections.

Phase I study of continuous-infusion recombinant macrophage colony-stimulating factor in patients with metastatic melanoma.

Macrophage colony-stimulating factor (M-CSF) is a lineage-specific, homodimeric growth factor that supports the proliferation and maturation of bone marrow progenitors and the survival and function of mononuclear/macrophage cells. In vitro studies have demonstrated antitumor activity of macrophage colony-stimulating factor-treated monocytes against melanoma target cells. A Phase I study was conducted by administering the glycosylated form of the protein to patients with metastatic melanoma as two 7-day continuous i.v. infusions separated by a 2-week rest. Cohorts of three patients per dose level received escalating doses of 10-160 microgram/kg/day. Safety, clinical, and biological effects were evaluated. The infusions were well tolerated with occasional maximum grade 2 nonhematological toxicity. Rapidly reversible thrombocytopenia was the major hematological adverse effect. Its etiology may in part be explained by proliferation and activation of monocyte/macrophage cells in bone marrow samples. Evidence for a biological effect on tumors was suggested by the delayed, complete disappearance of multiple lesions in one patient and a decrease in the size of one marker lesion in a second patient with a mixed response. Fasting serum cholesterol levels decreased during the infusions and may represent an additional therapeutic application for this growth factor.

Phase I trial of subcutaneous recombinant macrophage colony-stimulating factor: clinical and immunomodulatory effects.

PURPOSE: Recombinant human macrophage colony-stimulating factor (rM-CSF) has been demonstrated to control the growth, differentiation, and function of mononuclear phagocytes. Preclinical studies have indicated antitumor effects, and therefore a phase I trial of rM-CSF in patients with malignancy was initiated. The toxicity and hematologic and immunologic effects were investigated. PATIENTS AND METHODS: rM-CSF was administered as a subcutaneous injection on days 1 through 5 and 8 through 12. Cycles were repeated every 28 days. Cohorts of four to seven patients received rM-CSF at dose levels from 0.1 to 25.6 mg/m2/d. Forty-two patients received 88 cycles of rM-CSF. All patients had metastatic solid tumors refractory to standard therapy. RESULTS: The toxicity of rM-CSF was mild. Dose-limiting toxicity included thrombocytopenia (two patients) and iritis (one patient) occurring at a dose of 25.6 mg/m2/d. Hematologic studies demonstrated dose-related monocytosis occurring routinely at doses > or = 3.2 mg/m2/d, and thrombocytopenia. Immunologic studies demonstrated enhanced secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1 beta) by monocytes after in vitro stimulation with lipopolysaccharide, and increased expression of TNF-alpha mRNA at higher rM-CSF dose levels. Pharmacokinetic studies demonstrated that the systemic clearance rate of M-CSF increases during week 1 of therapy, resulting in lower blood levels of M-CSF during the second week of therapy. CONCLUSION: rM-CSF can be safely administered to patients, and has biologic activity on peripheral-blood monocytes.

Nonlinear pharmacokinetics of recombinant human macrophage colony-stimulating factor (M-CSF) in rats.

The pharmacokinetics and mechanisms of elimination of recombinant human macrophage-colony stimulating factor (M-CSF) were investigated in rats. Intravenous injections of 0.1, 1 or 10 mg/kg M-CSF were administered and plasma samples were measured for M-CSF by bioassay. Systemic clearance decreased and the shape of the concentration-time curve changed with increasing dose, indicating nonlinear pharmacokinetics. At 10 mg/kg, two half-lives were initially observed, but after about 20 hr the plasma M-CSF suddenly declined with a steep slope. The rapidly declining phase suggested a saturable clearance mechanism that was prominent at low plasma concentrations of M-CSF (below 300 ng/ml) and obscured at high plasma concentrations of M-CSF. The rapid decline of plasma M-CSF occurred at earlier times with multiple daily injections of M-CSF, indicating induction of the saturable clearance mechanism. The rapidly declining phase was inhibited by carrageenan, indicating that saturable clearance might be due to metabolism of M-CSF by macrophages. With ligation of either the renal pedicles or ureters, the apparent half-lives of M-CSF increased by a factor of 2- to 3-fold, while the occurrence of the rapidly declining phase was delayed, but not eliminated. Overall, the results are well described by a two-compartment, first-order elimination model with a parallel Michaelis-Menten elimination pathway. First-order elimination is largely performed by the kidneys and the saturable Michaelis-Menten elimination pathway appears to be mediated by cells of the monocyte-macrophage lineage.

Phase I trial of recombinant macrophage colony-stimulating factor by rapid intravenous infusion in patients with cancer.

Fourteen patients were entered into a phase I dose-escalation trial of macrophage colony-stimulating factor (M-CSF). M-CSF was administered to inpatients by rapid 15 min i.v. infusion every 8 h x 5 days, repeated after a 9-day rest. Dose levels evaluated were 20, 40, 80, 330, and 1,100 micrograms/m2. Monitoring of patients every 4 h included vital signs, daily complete blood count (CBC), and serum chemistries (SGOT, creatinine, and bilirubin) while receiving M-CSF. No clinical or laboratory evidence of toxicity was seen. The average serum t1/2 varied with dose level. At 330 and 1,100 micrograms/m2, the serum t1/2 was 25 and 84 min, respectively, implying a saturable mechanism of clearance. After 5 days of treatment, the t1/2 decreased by twofold, consistent with enhancement of the saturable mechanism. Monocyte cytotoxicity against the A375 melanoma cell line was evaluated pretreatment and day 5 of each cycle. No consistent enhancement of monocyte cytotoxicity was seen. No effect on peripheral blood monocyte number was seen until the 1,100 micrograms/m2 dose level. At this dose level, the mean monocyte number on day 5 was increased compared to baseline (1,300 mm3 vs. 300/mm3). Clinical activity was seen in two patients with previously progressive leiomyosarcoma metastatic to the liver. A partial response (PR) lasting 7 months occurred at the 330 micrograms/m2 dose level while a patient treated at 1,100 micrograms/m2 has had stable disease for 20+ months. The maximum tolerated dose (MTD) of M-CSF was not determined. Based on clinical responses, a phase II trial is warranted in patients with metastatic soft tissue sarcoma.

The effect of macrophage colony-stimulating factor on haemopoietic recovery after autologous bone marrow transplantation.

Macrophage colony-stimulating factor (M-CSF) is active in the late stages of monocyte maturation, activates mature monocyte-macrophages and enhances their production of various other cytokines. We have examined the effects of a 21 d course of escalating doses of M-CSF purified from human urine (hM-CSF) on recovery following autologous bone marrow transplantation (ABMT) in 20 patients with malignant lymphomas. Four patients were treated at each dose level of 4, 8, 16, 32 and 64 x 10(6) U/m2/d and results compared to 46 concurrent controls. There was no significant difference in recovery to an absolute neutrophil count (ANC) of 0.5 x 10(9)/l (median 20 d in hM-CSF group versus 22 in controls) or in recovery of platelets to 50 x 10(9)/l (32 d versus 39 d, 0.05 less than P less than 0.1); hM-CSF patients received a median of 81 platelet units following ABMT (controls 112 units, P = NS). hM-CSF patients had a median of 5.5 d with fever greater than 37.5 degrees C (control 8, P = NS), received parenteral antibiotics for 14.5 d (control 17, P = NS) and had a 50% incidence of bacteraemia (control 48%). hM-CSF treated patients were discharged by a median of day 29 following transplantation (control 33, P less than 0.05). Platelet and neutrophil recovery correlated significantly with the number of marrow mononuclear cells (MNC) reinfused in the hM-CSF group (P = 0.05 and P = 0.014 respectively) but not in controls. Subgroup analysis showed that hM-CSF patients receiving greater than 2 x 10(8) MNC/kg body weight reached an ANC of 0.5 x 10(9)/l by a median of day 16.5 (control 18.5, NS), became platelet transfusion independent by day 17 (control 29, P less than 0.05) and reached a platelet count of 50 x 10(9)/l by day 21 (control 40, P less than 0.05). No significant toxicity attributable to hM-CSF treatment was seen. These results suggest that hM-CSF accelerates platelet recovery following ABMT and that relatively large marrow innocula are required to see this effect.

A phase I trial of recombinant human macrophage colony-stimulating factor by rapid intravenous infusion in patients with refractory malignancy.

Twenty patients with advanced cancer for which there was no effective standard therapy or whose disease was refractory to standard therapy were treated with recombinant macrophage colony-stimulating factor (rM-CSF). The rM-CSF was administered by intravenous bolus infusion for 5 consecutive days every other week for 2 treatment weeks. The doses administered ranged from 30 to 33,000 micrograms/m2/day. There was no intrapatient dose escalation. There were minimal to no systemic side effects seen, except for acute dyspnea noted in three patients. The dyspnea was felt to be related to the rate of infusion and did not recur in one patient given additional rM-CSF at a slower infusion rate. The major hematologic effect seen was a mild decrease in platelet count, which began to recover while the patients continued to receive the rM-CSF. The clearance of rM-CSF was dose dependent. Lower doses resulted in a saturable mechanism felt to represent cellular uptake. Clearance at higher doses demonstrated both a first-order mechanism at high serum rM-CSF concentrations, representing renal clearance, as well as a saturable mechanism at low serum concentrations. The maximum mean serum half-life was reached at dose levels of greater than or equal to 3,690 micrograms/m2 and was in the range of 234-258 min. By this route of administration, rises in absolute monocyte count were slight and seen only at doses of greater than or equal to 450 micrograms/m2 during the second therapy week. The maximum tolerated dose was not reached in this study because of lack of availability of rM-CSF.

Alterations in the expression of colony-stimulating factor-1 and its receptor during an acute graft-vs-host reaction in mice.

During the course of an acute graft-vs-host reaction in the mouse we observed a progressive increase in the concentration of CSF-1 in serum and liver, peaking at day 14. In contrast, there was a progressive decrease in the splenic CSF-1 concentration. In vivo studies of 125I-CSF-1 uptake and degradation and in vitro studies of 125I-CSF-1 binding by splenic cells demonstrated that within 24 h of the reaction the number of CSF-1 receptor+ cells had increased by 2-fold and their capacity to express the CSF-1 receptor by approximately 3-fold, resulting in a approximately 2.5-fold increase in the splenic clearance of CSF-1 from the circulation. Inasmuch as at 24 h, serum CSF-1 was not significantly altered, these results are suggestive of an increased rate of release of CSF-1 into the circulation early in the response. The splenic CSF-1-receptor bearing cells were in a Mac-1+ fraction that is consistent with a role for CSF-1 in the generation of host-derived splenic macrophages in acute graft-vs-host reaction.