Protective, restorative, and therapeutic properties of recombinant colony-stimulating factors

Pretreatment of mice with recombinant murine (rM) colony-stimulating factor-granulocyte-macrophage (CSF-gm) or recombinant human (rH) CSF-g provides partial protection from the lethal effects of ionizing radiation or the alkylating agent cyclophosphamide (CTX). In addition, these agents can significantly prolong survival if administered following lethal doses of irradiation or CTX. To induce protective activity, cytokines were injected 20 hours before lethal irradiation or CTX administration. To accelerate recovery from lethal irradiation, the cytokines must be administered shortly following irradiation, and the induction of maximal levels of activity is dependent on chronic administration. In contrast, because of their longer half-lives, accelerated recovery from alkylating agents requires a delay of at least 24 to 48 hours to allow complete clearance of CTX before administration of a CSF. Studies quantitating peripheral blood leukocytes and bone marrow cellularity as well as colony-forming units per culture (CFU-C) frequency and CFU-C per femur revealed a significant correlation between these parameters and the ability to survive lethal irradiation. This is a US government work. There are no restrictions on its use.     

Interleukin 1-induced sequential myelorestoration: dynamic relation between granulopoiesis and progenitor cell recovery in myelosuppressed mice.

The myelorestorative effect of recombinant human interleukin 1 alpha (IL-1 alpha) was studied in mice treated with anticancer drugs. The treatment of mice with 5-fluorouracil (5-FU; 250 mg/kg body weight) or cyclophosphamide (CPA; 100 mg/kg) considerably decreased bone marrow or splenic colony-forming units in culture (CFU-C) or neutrophils in blood. The daily administration of IL-1 alpha (100 ng/mouse/day) after 5-FU treatment markedly accelerated the recovery of bone marrow CFU-C. This increase was followed by the recovery of splenic CFU-C and neutrophils in blood. In the CPA-treated mice the recovery of bone marrow CFU-C over the normal level was observed regardless of the administration of IL-1 alpha 3 days after CPA treatment. The daily administration of IL-1 alpha after CPA treatment markedly increased splenic CFU-C or neutrophils over the normal level following the increase of bone marrow CFU-C. Thus, in both 5-FU- and CPA-treated mice, the increase of bone marrow CFU-C after the administration of IL-1 alpha was observed several days earlier than the increase of splenic CFU-C and neutrophils in blood. The increase of splenic CFU-C and neutrophils in blood was observed concomitantly. The experiments, in which effective timing of IL-1 alpha injection was examined, indicated that the administration of IL-1 alpha within a few days after treatment with anticancer drugs was necessary for the accelerated recovery of bone marrow progenitor cells. On the other hand, the effective recovery of splenic progenitor cells and peripheral neutrophils required the administration of IL-1 alpha after the increase of bone marrow progenitor cells. Thus, the administration of IL-1 alpha daily or every other day was the most effective for recovery from myelosuppression induced by anticancer drugs.     

Copper(II)2(3,5-diisopropylsalicylate)4 stimulates hemopoiesis in normal and irradiated mice

We have previously reported that copper(II)2(3,5-diisopropylsalicylate)4 (Cu-DIPS) significantly increased the survival rate of mice exposed to lethal irradiation. To examine whether Cu-DIPS affected hemopoietic activity, groups of mice were treated with Cu-DIPS or vehicle and assayed for in vitro interleukin 3 (IL-3)-dependent colony-forming units (CFU-C) and for committed progenitor granulocyte-macrophage CFU (GM-CFU). Cu-DIPS increased the number of splenic IL-3 CFU-C by five- to sixfold 7 days after treatment and splenic GM-CFU by 12-fold on day 24. These increases were accompanied by a 50% increase in spleen weight. Bone marrow IL-3 CFU-C and GM-CFU were not affected at 7 or 14 days after treatment, but were somewhat depressed at 24 days. In irradiated (8.0 Gy) mice treated with Cu-DIPS or vehicle, splenic IL-3 CFU-C and GM-CFU were undetectable 7 days after irradiation, but recovered more rapidly in Cu-DIPS-treated mice. By 24 days splenic IL-3 CFU-C in Cu-DIPS-treated mice recovered to 150% of normal (unirradiated) values and GM-CFU recovered to 270% of normal, whereas irradiated control values remained at 25% and 7%, respectively. The recovery of bone marrow hemopoiesis was slower than spleen, but 42 days after irradiation Cu-DIPS-treated mice had higher levels of bone marrow IL-3 CFU-C (eightfold) and GM-CFU (4.6-fold) than vehicle-treated mice. Cu-DIPS stimulated sixfold increases in renewable, pluripotent stem cells as measured by the in vivo assay of endogenous colony-forming units (CFU-Se).     

Result of attempted hematopoietic reconstitution using isologous, peripheral blood mononuclear cells: a case report

In order to test the effect of peripheral blood mononuclear cell infusions on hematopoietic recovery in man we intensively leukapheresed a normal identical twin and obtained 9.8 x 10(10) peripheral blood mononuclear cells containing 4 x 10(5) CFU-C. These isologous cells were infused into his identical twin brother who had received 150 rad of total body irradiation and intensive combination chemotherapy as adjuvant therapy for Ewing's sarcoma. When compared to other patients receiving similar treatment, leukocyte recovery was accelerated by 3-4 wk, and occurred at a rate comparable to that induced by infusion of autologous cryopreserved marrow. Recovery of granulocytes, monocytes and platelets was not accelerated. The low number of CFU-C present in the preparation used ((one-eighth the number of CFU-C we usually obtain from bone marrow autograft collections) may have led to the pattern of hematopoietic recovery we observed in this patient.     

Granulocyte-macrophage progenitor cell preservation at 4°C

Eighteen bone marrows collected from patients without haematological diseases and from normal subjects were tested for the effects of 4 d storage at 4 degrees C on CFU-C growth. Results indicate that unfractionated bone marrow cells may be stored at 4 degrees C for 4 d with 97% +/- 8 SEM recovery of the CFU-C evaluated by the agar culture assay. On the other hand, the same preservation procedure on peripheral blood CFU-C of 13 normal subjects yielded only 5% +/- 2 SEM recovery of in vitro growth capacity. The present results have practical implications. They might be exploited to preserve bone marrow CFU-C for transplantation therapy or laboratory investigation. In contrast this single preservation procedure seems not appropriate for preserving blood CFU-C.     

Studies on the regeneration of the CFU-C population in blood and bone marrow of lethally irradiated dogs after autologous transfusion of cryopreserved mononuclear blood cells

In a group of 8 lethally irradiated (1200 R) dogs, that were transfused autologously with cryopreserved mononuclear cells (MNC) derived from the peripheral blood by leucapheresis the concentration of colony-forming units in agar (CFU-C) in bone marrow and peripheral blood was estimated at regular intervals after irradiation and transfusion of MNC. The numbers of MNC transfused per kg body weight ranged from 0.32 x 10(9) to 1.63 x 10(9) with an incidence of CFU-C between 0.02 x 10(5) and 1.38 x 10(5). In 6 dogs the CFU-C levels in the bone marrow reached the normal pre-irradiation values between days 15 and 20. But in 2 dogs that had received the lowest CFU-C numbers the regeneration of the bone marrow CFU-C was markedly delayed. In general the time course of the bone marrow repopulation by CFU-C for single dogs was reflected by a corresponding regeneration pattern of the blood CFU-C. The time course of the curves for the blood CFU-C levels on the other hand was of the same kind as for the granulocyte values in the peripheral blood, thuations were seen in the blood CFU-C levels of single dogs before irradiation and after mononuclear leucocyte transfusion. Despite of such limitations the blood CFU-C content appeared to be a useful indicator of haematopoietic regeneration of the bone marrow.     

The role of drugs and lymphocytes in granulocyte-macrophage colony formation in patients with drug induced agranulocytosis

The in vitro effects of the causative drugs and lymphocytes from the patients with agranulocytosis were tested against granulocyte-macrophage colony formation (CFU-C) of bone marrow cells from normal individuals and the patients in recovery stage. A semisolid culture system was used for CFU-C assay. The drug concentrations were adjusted to the therapeutic levels in sera, and the lymphocytes were obtained from the patient's peripheral blood. Three patients with agranulocytosis and one patient with pancytopenia caused by disopyramide, methimazole, sodium valproate, and Towasaal, respectively, were examined. Each of the four drugs except disopyramide suppressed the CFU-C of normal and patient's bone marrow cells in a dose-dependent manner. When the patient's bone marrow cells were cultured with respective drugs and their own lymphocytes or with the culture supernatant of the drug and lymphocytes, CFU-C suppressions was significantly augmented. Phenacetin, an agent of Towasaal, significantly suppressed CFU-C and also CFU-E. These results indicate that humoral factor(s) produced from patient's lymphocytes by reacting with the drugs may function as an immunological mechanism in the patients with drug-induced agranulocytosis.     

CFU-C Populations in Blood and Bone Marrow of Dogs after Lethal Irradiation and Allogeneic Transfusion with Cryopreserved Blood Mononuclear Cells

Colony forming units in agar (CFU-C) were assayed in both bone marrow and peripheral blood of dogs during haemopoietic recovery after lethal total-body irradiation (1200 R) and allogeneic transfusion of blood mononuclear cells (MNC) from histocompatible donors. MNC had been collected from the peripheral blood by continuous-flow centrifugation leucapheresis and cryopreserved at -196°C until transfusion. Two groups of dogs were studied. Group 1 dogs (n = 12) were given between 0.39 and 2.76 times 10⁹ MNC per kg body wt. Group 2 dogs (n = 14) were transfused with a similar number of MNC, ranging from 0.51 to 1.87 times 10⁹ per kg body wt., but in addition underwent immuno-suppressive therapy with methotrexate. In group 1 dogs, there was a rather good correlation between the number of CFU-C in the regenerating bone marrow and the recovery of the peripheral blood granulocyte values. The regeneration of the CFU-C population in the bone marrow of methotrexate-treated dogs showed a somewhat more heterogeneous picture than in dogs of group 1 and in dogs that, in a previous study, were transfused with autologous MNC. The minimum time interval required for the reconstitution of peripheral blood CFU-C to normal levels was 2–4 weeks but usually took from 4–14 weeks.     

Abnormal responses of myeloid progenitor cells to recombinant human colony-stimulating factors in congenital neutropenia.

The effects of recombinant human interleukin-3 (IL-3) and recombinant human granulocyte colony-stimulating factor (G-CSF) on the growth of myeloid progenitor cells (CFU-C) in semisolid agar culture were studied in two patients with Kostmann-type congenital neutropenia. CFU-C growth in bone marrow cells from patients was significantly reduced in response to various concentrations of either IL-3 or G-CSF alone, compared with that from normal subjects. There was no inhibitory effect of bone marrow cells from patients on normal CFU-C formation supported by IL-3 or G-CSF. However, the simultaneous stimulation with IL-3 and G-CSF induced the increase of CFU-C formation in patients with congenital neutropenia. Furthermore, CFU-C growth in both patients was supported when bone marrow cells were preincubated with IL-3 in liquid culture followed by the stimulation with G-CSF in semisolid agar culture. In contrast, that was not supported by the preincubation with G-CSF and the subsequent stimulation with IL-3. This evidence suggests that the hematopoietic progenitor cells in patients with congenital neutropenia have the potential for developing CFU-C in the combined stimulation with IL-3 and G-CSF, and that this growth may be dependent on the priming of IL-3 followed by the stimulation with G-CSF. The level of mature neutrophils in peripheral blood was not fully restored to normal levels by the daily administration of G-CSF in doses of 100 to 200 micrograms/m2 of body surface area for 20 to 25 days in both patients. These observations raise the possibility that the combination of IL-3 and G-CSF might have a potential role for the increase of neutrophil counts in patients with congenital neutropenia.     

CCNU Toxicity After an Overdose in a Patient with Hodgkin's Disease

An overdose of CCNU (600 mg over a 15-d period) was unintentionally ingested by a patient with advanced Hodgkin's disease subjected to combination chemotherapy. A severe bone marrow depression occurred 3 weeks after the start of the CCNU treatment. The nadir of the platelet count was reached after 4 weeks and that of the granulocyte count after 5 weeks. At the nadir of the white blood cell count, colony-forming cells (CFU-C) were found in significantly reduced numbers in the bone marrow, and were not found at all in the peripheral blood; the amount of colony-stimulating activity (CSA) produced by peripheral blood cells was reduced. However, the cells producing CSA recovered earlier than the CFU-C, and the CSA peak value was reached about 1 week before the peak value for CFU-C in the bone marrow. Thus, in vivo CSA-producing cells appeared to be more resistant to CCNU than were CFU-C, and their recovery appeared to be a prerequisite for the recovery of CFU-C and myelopoietic cells.     

Effects of recombinant granulocyte colony-stimulating factor (rG-CSF) and recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) on acute radiation hematopoietic injury in mice

We have attempted to evaluate in vivo effects of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on acute radiation hematopoietic injury in mice. BDF1 mice, irradiated with 7.5-Gy x-rays, were injected i.p. twice daily for 10 days with 10(5) U recombinant human G-CSF, 3.75 x 10(5) U recombinant murine GM-CSF, or a combination of both. G-CSF significantly enhanced the recovery of not only peripheral leukocytes but also platelets and hematocrit on days 14 and 21 after irradiation. GM-CSF significantly enhanced the recovery of platelets on day 14 and peripheral leukocytes on day 21. G-CSF markedly enhanced the recovery of spleen colony-forming units (CFU-S), colony-forming units in culture (CFU-C), erythroid burst-forming units (BFU-E), and megakaryocyte colony-forming units (CFU-Meg) both in bone marrow and in the spleen. GM-CSF significantly enhanced the recovery of CFU-Meg in bone marrow on day 14. We found synergistic effects between G-CSF and GM-CSF on CFU-S, CFU-C, and CFU-Meg in the spleen on day 14, although we found antagonistic effects between G-CSF and GM-CSF on CFU-S and CFU-C in bone marrow on day 7, and on platelet counts on day 7. These results indicate that the administration of recombinant G-CSF and GM-CSF may be useful in accelerating hematopoietic recovery in patients with acute radiation hematopoietic injuries.     

Hematopoietic stem cell depletion by restorative growth factor regimens during repeated high-dose cyclophosphamide therapy.

We studied the effects of six cycles of repeated cyclophosphamide (CTX) therapy followed by restorative therapy with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF on the hematopoietic stem cell compartment. Stem cell function was assessed by serially transferring bone marrow cells from CTX-CSF-treated mice into lethally irradiated recipient mice. Bone marrow cells from mice that initially received either G-CSF or GM-CSF after CTX therapy more rapidly lost the ability to repopulate the recipient lymphoid organs, showed a dramatic loss of hematopoietic progenitors, a more rapid loss of CFU-S capacity, and a 40% to 50% reduction in marrow repopulating ability (MRA). Interleukin-1 (IL-1) appeared to have little effect on the CTX-treated mice when used alone. However, when administered before the CTX-CSF regimen, IL-1 prevented the stem cell depletion as determined by CFU-C, CFU-S, and MRA through the serial transplantation procedures. These results support the hypothesis that repeated treatments with myelosuppressive drugs followed by stimulation with the CSFs may induce damage to the host stem cell compartment, and further suggest that pretreatment with IL-1 before CTX therapy may prevent this stem cell damage.     

Enhancement of hemopoietic recovery after bone marrow transplantation with the addition of nucleated blood cells in mice

Recovery of bone marrow cellularity, CFU-C, and CFU-S were studied sequentially over 90 days time after syngeneic bone marrow transplantation in mice. A minimal cell dose of 2 X 10(5) bone marrow cells was given. At day 28 after transplantation, CFU-C reached more than 50% of the normal range whereas the CFU-S concentration was less than 15%. Normalization of CFU-S occurred at day 90. The effect of the addition of peripheral blood nucleated cells on bone marrow hemopoietic recovery was studied at day 28. The augmentation of CFU-C and CFU-S recoverey were dose dependent. Optimal enhancement was seen with bone marrow to blood ratios of 1:1 and 1:2.5. This enhancement effect was lost when nucleated blood cells in a ratio of 1:10 were administered.     

In Vitro Colony Studies in 87 Patients with Acute Nonlymphoblastic Leukemia

ABSTRACT. Colony-forming cells (CFU-C) in peripheral blood and bone marrow and colony-stimulating activity (CSA) in mononuclear peripheral white blood cells were studied at diagnosis in 87 patients with acute nonlymphoblastic leukemia (ANLL). Absence of CFU-C in peripheral blood was more frequent in patients who did not enter remission than in those who did, and survival was significantly shorter in CFU-C-negative than in CFU-C-positive patients. No correlation was found between CFU-C in the bone marrow and frequency of remission or survival time. Absence of CSA was significantly more frequent in patients who did not enter remission than in those who did. Only 4 of 28 patients who lacked CSA entered remission. Survival was significantly longer in CSA-positive than in CSA-negative patients. Thus, CSA synthesis in peripheral mononuclear blood cells appears to be a valuable prognostic factor in ANLL.     

Recovery of normal hematopoietic tissue and tumor following chemotherapeutic injury from cyclophosphamide (CTX): comparative analysis of biochemical and clinical techniques

Simultaneous alterations in the incorporation of 3H-thymidine (3H-TdR) into DNA are induced by CTX in normal host target tissues and L1210 ascites tumor. The timing of suppression and recovery of these nucleoside incorporation alterations was similar at the three CTX doses studied, but some evidence for a dose-response effect was seen as the magnitude of suppression of DNA synthesis increased with increasing dosage. A differential pattern of suppression and recovery of 3H-TdR incorporation in malignant and normal host tissues was observed. The pattern of suppression and recovery of the peripheral white blood count and bone marrow (BM) cellularity, two frequently studied clinical parameters of hematopoietic recovery, were out of phase with the recovery of BM-DNA synthesis and failed to accurately reflect the sensitivity of the BM to subsequent chemotherapeutic injury. In contrast, drug schedules based on the differential recovery patterns of the host tissues and tumor, reflected by their 3H-TdR incorporation into DNA, both reduced toxicity to normal mice and increased the survival of tumor-bearing animals.     

Granulopoiesis in severe congenital neutropenia.

The pathogenesis of the granulopoietic failure in three children with severe congenital neutropenia was studied. Mature neutrophils were absent from both peripheral blood and bone marrow. Assay of bone marrow granulocyte colony-forming cells (CFU-C) in a methylcellulose tissue culture system using colony-stimulating activity (CSA) from peripheral blood leukocytes demonstrated normal or increased concentrations of CFU-C compared to those from marrows of 60 age-matched controls. Colonies were of normal size and by light microscopy appeared to contain granulocytes in all stages of maturation including the mature polymorphonuclear neutrophil. CFU-C from peripheral blood of two patients were normal. Production and activity of CSA from the patients' peripheral blood leukocytes and urinary CSA excretion were normal. No serum inhibitors against CFU-C or CSA could be demonstrated using both control and autologous marrow. The defect did not appear to be due to a lack of granulocytic stem cells, a reduction of humoral stimulators of granulopoiesis, nor the presence of an inhibitor as measured by these techniques.     

Increased Levels of Myeloid Progenitor Cells in the Blood of Patients with Metastatic Invasion of the Marrow

Granulocyte-macrophage clusters and colony-forming cells (CFU-C) in the peripheral blood have been studied in 26 cancer patients with neoplastic bone marrow involvement. The concentration of CFU-C in the blood of normal individuals and of cancer patients with no bone marrow invasion, ranged from 0 to 99 ml. In contrast, out of 27 cancer patients with marrow invasion, 9 (35%) showed a significant increase of blood CFU-C (100 to 21000/ml) and of those 5 (19%) showed an increase of blood colonies (41 to 9000/ml). There was a strong correlation between increased CFU-C or colony concentration and the presence of myeloid or/and erythroid immature cells in the peripheral blood. On the other hand, there was no apparent correlation between an increased CFU-C level and anaemia or abnormal blood leucocyte count or marrow fibrosis. These observations suggest that bone marrow involvement by neoplastic cells may cause spatial redistribution of the grnulocyte macrophage progenitor cells.     

Proliferative state of normal in vitro colony-forming cells during development of L5222 rat leukemia and their reaction to chemotherapy

In the experimental rat leukemia, L5222, the decrease of normal in vitro colony-forming cells (CFU-C) after chemotherapy with daunomycin is much less than in nonleukemic controls. The leukemia is therefore used here to test the hypothesis that in leukemia the CFU-C are expelled from the active cell cycle to a resting state and are thereby less sensitive to cycle-dependent chemotherapeutic agents. The L5222 leukemia has the advantage that the leukemic blast cells do not form colonies in agar culture so that normal CFU-C can be assessed under leukemic conditions. To compare the proportions of CFU-C in the S-phase in normal and leukemic rats, two S-phase-specific agents, 3H-thymidine and hydroxyurea, were used to kill proliferating bone marrow cells. Following treatment with 3H-thymidine in vitro, about 41% of the CFU-C were killed in normal and about 25% in leukemic bone marrow. Hydroxyurea administered in vivo resulted in the death of about 33% and 26%, respectively. The results indicate that fewer normal CFU-C are in S-phase in the L5222 leukemia, which might help to explain how enough normal stem cells survive chemotherapy to regenerate the bone marrow.     

Increase in circulating stem cells following chemotherapy in man.

The number of circulating granulocytic stem cells (CFU-C) was determined by the in vitro methylcellulose technique in cancer patients receiving intermittent chemotherapy. In 17 patients studied prior to therapy, the median CFU-C concentration per 2 X 10(5) mononuclear cells plated was six, compared to a posttreatment median of 23 in 21 patients (p less than 0.001). Large numbers of stem cells were obtained by leukopheresis and cryopreserved with a 99.5% median CFU-C recovery. Cyclical changes in the concentration of stem cells with maximum values of 20 times baseline were demonstrated in a patient studied at weekly intervals during multiple courses of treatment. It was estimated that, at peak CFU-C concentrations, a quantity of stem cells equivalent to that present in a bulk bone marrow harvest could be obtained from the peripheral blood by a 17-liter pheresis. These results suggest that it may be practical to obtain an adequate number of stem cells from the peripheral blood to study autologous stem cell infusion as a means of averting myelosuppression in patients receiving intensive chemotherapy.     

Interleukin-1 administration before lethal irradiation and allogeneic bone marrow transplantation: early transient increase of peripheral granulocytes and successful engraftment with accelerated leukocyte, erythrocyte, and platelet recovery

Administration of interleukin-1 beta (IL-1 beta) before a lethal irradiation with or without allogeneic bone marrow transplantation (BMT) protects greater than 90% of the irradiated mice. To approach the mechanisms responsible for the radioprotective effect of IL-1, we examined the effects of IL-1 pretreatment on engraftment and kinetics of peripheral blood, spleen, and marrow cell reconstitution after irradiation and BMT. Although the BMT was not necessary for the survival of the IL-1-pretreated lethally irradiated mice, allogeneic marrow did engraft in these mice as evaluated in the spleen and marrow 2 months after BMT. IL-1 pretreatment significantly accelerated hematopoietic recovery versus transplanted saline-treated controls with a pronounced enhancement of peripheral leukocyte, platelet, and erythrocyte recovery. Leukocyte recovery in IL-1-pretreated mice was unique in that IL-1 first induced an early transient (maximum at day 7) increase of peripheral granulocytes before accelerating leukocyte recovery after day 11. IL-1 pretreatment also significantly enhanced marrow cell recovery after allogeneic BMT with an eightfold increase in marrow cellularity from day 4 to 11 versus control transplanted mice. When lethal irradiation was not followed by allogeneic BMT. IL-1 pretreatment also affected the peripheral reconstitution of leukocytes, platelets, and erythrocytes. Interestingly, in the absence of BMT, IL-1 also induced an early circulation of peripheral granulocytes. Overall, our data demonstrate that a single administration of IL-1 before lethal irradiation and allogeneic BMT can induce an early transient increase of circulating granulocytes, followed by an accelerated multilineage recovery and long-term allogeneic engraftment.