Most primitive hematopoietic stem cells are stimulated to cycle rapidly after treatment with 5-fluorouracil

To test whether primitive hematopoietic stem cells (PHSC) cycle rapidly during recovery from an initial 5-fluorouracil (5-FU) treatment, two doses of 5-FU were administered 1, 3, 5 or 8 days apart. Cells from treated marrow donors were mixed with untreated competitor marrow that would produce genetically distinguishable erythrocytes and lymphocytes, using hemoglobin (Hb) and glucosephosphate isomerase (GPI) transplantation markers. These cell mixtures were injected into lethally irradiated hosts. Functional abilities of donor marrow populations were assessed after 3, 6, and 12 months as percentages of donor type Hb and GPI in the host's circulating erythrocytes and lymphocytes, respectively. Bone marrow from mice treated with two doses of 5-FU 3 to 5 days apart was severely affected, producing circulating erythroid and lymphoid cells an average of only 25% of normal for doses 3 days apart, and 14% of normal for doses 5 days apart. Two doses of 5-FU administered 1 day or 8 days apart had much smaller effects, producing circulating cells 75% or 58% of normal. Thus, most PHSC are stimulated to proliferate rapidly 3 to 5 days after treatment with 5-FU, but far fewer PHSC proliferate as early as 1 day, or as late as 8 days, after the 5-FU treatment.     

Local irradiation enhances congenic donor pluripotent hematopoietic stem cell engraftment similarly in irradiated and nonirradiated sites

Long-term multilineage chimerism is achieved in CD45 congenic mice receiving high bone marrow doses with or without mediastinal irradiation (MI). Increased donor chimerism results in MI-treated compared with nonirradiated animals, suggesting that MI makes "space" for engraftment of donor pluripotent hematopoietic stem cells (PHSCs). We have now examined whether space is systemic or whether increased engraftment of donor marrow in locally irradiated mice is confined to the irradiated bones. While increased donor chimerism was observed in irradiated bones compared with nonirradiated bones of MI-treated animals 4 weeks following bone marrow transplantation (BMT), these differences were minimal by 40 weeks. MI-treated chimeras contained more adoptively transferable donor PHSCs in the marrow of both irradiated and distant bones compared with non-MI-treated chimeras. Similar proportions of donor PHSCs were present in irradiated and nonirradiated bones of locally irradiated mice at both 4 and 40 weeks. Irradiated bones contained more donor short-term repopulating cells than distant bones at 4 weeks, but not 40 weeks, after BMT. Our study suggests that local proliferation of donor PHSCs in mice receiving local irradiation rapidly leads to a systemic increase in donor PHSC engraftment.     

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on primitive hematopoietic stem cell (PHSC) function and numbers, after chemotherapy

OBJECTIVE: Primitive hematopoietic stem cell function was assessed after cyclophosphamide with granulocyte-macrophage colony-stimulating factor (GM-CSF), with or without preadministration of interleukin-1, using competitive repopulation. METHODS: C57B6/J mice injected with one or four biweekly intravenous injections of cyclophosphamide, 200 mg/kg, received granulocyte-macrophage colony-stimulating factor, 1 microg, subcutaneously for 5 days, beginning 24 hours after cyclophosphamide. Alternatively, mice were injected with interleukin-1, 1 microg, 20 hours before administration of drug or drug and cytokine. Marrow obtained from mice sacrificed 4 weeks after the last dose of drug or drug and cytokine was used in competitive repopulation. RESULTS: Significant reductions in marrow repopulating ability occurred after a single dose of cyclophosphamide or multiple injections. Repopulating units (RU) were calculated, and both binomial and Poisson models for estimation of primitive hematopoietic stem cell (PHSC) numbers were used. RU were significantly diminished for all treatment groups when compared to controls. PHSC numbers were not significantly affected by either regimen of cyclophosphamide given alone. Addition of GM-CSF to cyclophosphamide, whether the latter was given in single or multiple doses, led to further, although insignificant, declines in repopulating ability, as well as PHSC and RU numbers. Interleukin-1 usage exacerbated the observed repopulating defect. There was evidence of replicative failure in individual cells, indicating a qualitative defect also. SUMMARY: Additive stem cell depletion and qualitative replicative defect occur after chemotherapy-cytokine usage. However, the replicative defect of PHSC seen after addition of GM-CSF is not significantly worse than that seen with cytotoxic drug use alone.     

Identification of in vitro growth conditions for c-Kit-negative hematopoietic stem cells

Our laboratory recently identified a quiescent class of pluripotent hematopoietic stem cells (PHSCs) that are lineage negative (Linneg), lack c-Kit, and are able to give rise to c-Kit-positive (c-Kitpos) PHSCs in vivo. This population fails to proliferate in vitro but has delayed reconstituting activity in vivo. In this study, we purified these cells to enrich for the PHSCs and we identified in vitro conditions capable of supporting their maturation. The c-Kit-negative (c-Kitneg) cells exhibited differential expression of Sca-1, CD34, CD43, CD45, and Thy 1.2. We purified the cells based on Sca-1, as it is expressed on active PHSCs. We detected pre-colony-forming unit spleen (pre-CFU-s) activity in both the Sca-1neg and Sca-1pos populations, indicating the presence of primitive PHSCs in both populations. However, our in vitro studies suggest that the Sca-1pos population is enriched for PHSCs. The in vitro systems that support the growth of these dormant cells include a modified long-term marrow culture and various stromal cell lines. In modified long-term bone marrow cultures, c-Kitneg cells gave rise to c-Kitpos PHSCs, with long-term reconstitution activity in vivo. Thus we have established an in vitro system to examine PHSC maturation that will allow us to study the mediators of the c-Kitneg to c-Kitpos transition.     

Genetic regulation of primitive hematopoietic stem cell senescence

OBJECTIVE: To define effects of strain on PHSC (primitive hematopoietic stem cells) senescence (decline in function with age) in vivo, and to map a locus that regulates PHSC senescence. MATERIALS AND METHODS: Long-term function and self-renewal were compared in bone marrow cells (BMC) from old and young mice of three strains: BALB/cBy (BALB), DBA/2 (D2) and C57BL/6 (B6), using competitive repopulation and serial transplantation in vivo. BMC from each old or young donor were mixed with standard doses of congenic, genetically marked BMC and transplanted into lethally recipients. Percentages of donor-type erythrocytes and lymphocytes in the recipients determined the functional ability of donor PHSC relative to the standard, where one repopulating unit (RU) of donor BMC equals the repopulating ability of 100,000 standard competitor BMC. Using similar techniques, repopulating abilities of old and young recombinant inbred (RI) donors of 12 strains derived from BALB and B6 were compared in NK-depleted BALBxB6 Fl recipients to map a locus that appears to have a major role in PHSC senescence. RESULTS: PHSC function declined about 2 fold with age in BALB and D2 BMC, and increased more than 2-fold with age in B6 BMC, with all old/young strain differences significant, p<.01. Ten months after serial transplantation, young B6, BALB, and D2 PHSC had self-renewed 1.6-, 4.2-, and 3.2-fold better than old, with BALB and D2 old/young differences p<.01. Young B6 PHSC self-renewed 1.9- and 2.9-fold better than young BALB and D2 PHSC. The PHSC senescence phenotypes (old/young RU ratios) for 12 CXB RI strains suggested a genetic linkage to D12Nyul7 on Chromosome 12. CONCLUSION: PHSC senescence is genetically regulated, and is much delayed in the B6 strain compared to the BALB and D2 strains. A locus on Chromosome 12 may regulate PHSC senescence.     

Efficient retrovirus transduction of mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood by treatment with granulocyte colony-stimulating factor and stem cell factor

Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recovery after bone marrow (BM) transplantation, and are proposed to be useful targets for retrovirus- mediated gene transfer protocols. We treated mice with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to mobilize hematopoietic progenitor cells into the peripheral blood. These cells were analyzed for the number and frequency of pluripotent hematopoietic stem cells (PHSC). We found that splenectomized animals treated for 5 days with G-CSF and SCF showed a threefold increase in the absolute number of PHSC over normal mice. The number of peripheral- blood PHSC increased 250-fold from 29 per untreated mouse to 7,200 in peripheral-blood PHSC in splenectomized animals treated for 5 days with G-CSF and SCF. Peripheral blood PHSC mobilized by treatment with G-CSF and SCF were analyzed for their ability to be transduced by retroviral vectors. Peripheral-blood PHSC from splenectomized animals G-CSF and SCF were transduced with a recombinant retrovirus containing the human MDR-1 gene. The frequency of gene transfer into peripheral blood PHSC from animals treated for 5 and 7 days was two-fold and threefold higher than gene transfer into PHSC from the BM of 5-fluorouracil-treated mice (P < .01). We conclude that peripheral blood stem cells mobilized by treatment with G-CSF and SCF are excellent targets for retrovirus- mediated gene transfer.     

Engraftment of bone marrow cells into normal unprepared hosts: effects of 5-fluorouracil and cell cycle status

Bone marrow from animals treated with 5-fluorouracil (5FU) competes equally with normal marrow when assessed in vivo in an irradiated mouse, but shows markedly defective engraftment when transplanted into noncytoablated hosts. Using Southern Blot analysis and a Y-chromosome specific probe, we determined the level of engraftment of male donor cells in the bone marrow, spleen, and thymus of unprepared female hosts. We have confirmed the defective engraftment of marrow harvested 6 days after 5FU (FU-6) and transplanted into unprepared hosts and shown that this defect is transient; by 35 days after 5FU (FU-35), engraftment has returned to levels seen with normal marrow. FU-6 marrow represents an actively cycling population of stem cells, and we hypothesize that the cycle status of the stem cell may relate to its capacity to engraft in the nonirradiated host. Accordingly, we have evaluated the cycle status of engrafting normal and FU-6 marrow into normal hosts using an in vivo hydroxyurea technique. We have shown that those cells engrafting from normal marrow and over 70% of the cells engrafting from FU-6 marrow were quiescent, demonstrating no killing with hydroxyurea. We have also used fluorescent in situ hybridization (FISH) analysis with a Y-chromosome probe and demonstrated that normal and post-5FU engraftment patterns in peripheral blood were similar to those seen in bone marrow, spleen, and thymus. Altogether these data indicate that cells engrafting in normal, unprepared hosts are dormant, and the defect that occurs after 5FU is concomitant with the induction of these cells to transit the cell cycle.     

Alloantigen recognition is critical for CD8 T cell-mediated graft anti-tumor activity against murine BCL1 lymphoma after myeloablative bone marrow transplantation

The goal of the current study was to determine whether whole bone marrow cells or splenic CD8(+) T cells from C57BL/6 (H-2(b)) donor mice, which are tolerant to BALB/c (H-2(d)) alloantigens, are capable of mediating graft anti-tumor activity against a BALB/c B-cell lymphoma after injection into irradiated BALB/c hosts. The experimental results show that high doses of splenic CD8(+) T cells mixed with T cell-depleted bone marrow cells from C57BL/6 non-tolerant (normal) donors eliminate the BCL(1) B-cell lymphoma cells and induce lethal graft-versus-host disease (GVHD). CD8(+) T cells from tolerant donors simultaneously lose both their ability to induce GVHD and their anti-tumor activity. Whole bone marrow cell transplants from normal donors eliminated BCL(1) tumor cells without inducing GVHD, and bone marrow cells from tolerant donors failed to eliminate the tumor cells. The infused BCL(1) tumor cells expressed an immunogenic tumor-specific idiotype antigen disparate from host alloantigens, indicating that recognition of the tumor-specific antigen alone was insufficient to elicit graft anti-tumor activity from unimmunized allotolerant donor splenic CD8(+) T cells or whole bone marrow cells. We conclude that CD8(+) T cells from unimmunized normal donor mice require alloantigen recognition to mediate their anti-tumor activity following allogeneic BMT.     

Enhanced Retroviral Transduction of 5-Fluorouracil-Resistant Human Bone Marrow (Stem) Cells Using a Genetically Modified Packaging Cell Line

Pluripotent hematopoietic stem cells (PHSC) are rare cells capableof multilineage differentiation, long-term reconstituting activity andextensive self-renewal. Such cells are the logical targets for manyforms of corrective gene therapy, but are poor targets for retroviralmediated gene transfer owing to their quiescence, as retroviraltransduction requires that the target cells be cycling. To try andsurmount this problem we have constructed a retroviral producer linethat expresses the membrane-bound form of human stem cell factor (SCF)on its cell surface. These cells are capable, therefore, of deliveringa growth signal concomitant with recombinant retroviral vectorparticles. In this report we describe the use of this cell line totransduce a highly quiescent population of cells isolated from adulthuman bone marrow using the 5-fluorouracil (FU) resistance technique ofBerardi et al. Quiescent cells selected using this technique weretransduced by cocultivation with retroviral producers expressingsurface bound SCF or with the parent cell line that does not. Followingcoculture, the cells were plated in long-term bone marrow culture for afurther 5 weeks, before plating the nonadherent cells in semisolidmedia. Colonies forming in the semisolid media over the next 14 dayswere analyzed by polymerase chain reaction for the presence of theretroviral vector genome. Over six experiments, the transductionfrequency of the quiescent 5-FU resistant cells using theSCF-expressing producer line averaged about 20%, whereas thosetransduced using the parent producer line showed evidence of reducedlevels or no transduction.     

Influence of Antioxidants on the Quality of Stored Blood

Background and objectives: Blood is exposed to oxidation stress and therefore has a high antioxidant capacity (AOC). With the many factors increasing the demands on the AOC, there may be damage to erythrocytes by free radicals. This study was to investigate evidence of erythrocyte damage in stored donor blood and to affect this by premedication of blood donors. Materials and methods: Blood samples of 15 healthy donors were collected in CPDA-1 solution and analyzed immediately, and then again after 10 days of incubation at 4 °C and 1 day of incubation at 37 °C. Prior to incubation, the following parameters were evaluated: Na⁺, K⁺, malondialdehyde (MDA), hemoglobin (Hb), AOC in the supernatant, superoxide dismutase (SOD) in erythrocytes, and glutathione peroxidase (GSHPx) in whole blood. Blood donors of group 1 were not given any drugs or vitamins before blood sampling. The same blood donors were then supplemented with the following daily doses of antioxidants for 10 days before the next blood sampling: 36 mg of β-carotene, 300 mg of vitamin E, 200 mg of vitamin C, and 40 mg of selenium. Results: The blood from donors of group 2 had a significantly smaller increase in MDA, K⁺, and Hb, and a smaller decrease in Na⁺ and AOC in the supernatant compared with that of group 1, while the activity of SOD and GSHPx did not change during blood storage. Conclusions: These results suggest that antioxidants given to blood donors can improve red cell storage parameters by reducing cell damage caused by free radicals.     

Transforming growth factor beta 1 systemically modulates granuloid, erythroid, lymphoid, and thrombocytic cells in mice.

Transforming growth factor beta 1 (TGF-beta 1) has been shown to inhibit the development of most early hemopoietic progenitors in vitro. The present series of in vivo experiments show that TGF-beta 1 can simultaneously augment and suppress distinct cell lineages in peripheral and central hemopoietic compartments. Mice treated daily for 7-14 days with s.c. injections of TGF-beta 1 exhibited up to a 95% reduction in circulating platelets and a 50% reduction in red cell counts, whereas a 50%-400% increase occurred in circulating white cells with the morphology of small lymphocytes. Decreased erythrocytes were also evident in the splenic red pulp and bone marrow sinusoids. A dramatic increase in granulopoiesis occurred in the spleen and bone marrow, followed by a peripheral neutrophilia 1 week after treatments ceased. All effects were completely reversible, with normal histologic and hematologic profiles evident 2 weeks after cessation of treatments. Thus, TGF-beta 1 can differentially regulate multiple hemopoietic pathways in a systemic, reversible, and dose-dependent fashion. These actions may be mediated by the direct effects of TGF-beta 1 or through modulation of secondary cytokines and receptors.     

The effects of daily recombinant human granulocyte colony-stimulating factor administration on normal granulocyte donors undergoing leukapheresis [see comments]

The effects of daily administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to eight normal volunteers donating granulocytes for neutropenic relatives undergoing marrow transplantation were studied. Granulocyte donors consisted of seven marrow donors (5 syngeneic, 2 HLA identical) and one haploidentical son who had not donated marrow. All donors were administered daily rhG-CSF at a mean dose of 5 micrograms/kg/d (range 3.5 to 6.0) for a mean of 11.75 days (range 9 to 14 days), and granulocytes were collected a mean of 7.6 times (range 4 to 12). RhG-CSF was well tolerated and only minor side effects were observed. All donors became anemic from marrow donation and the removal of red blood cells during the collection procedures. Red blood cell transfusions were not given. All donors had a decrease in platelet counts and the magnitude of the decrement appeared to be greater than in historical donors. This was due in part to increased removal of platelets with the collection product, but a direct effect of rhG-CSF on platelet production cannot be excluded. The mean precollection granulocyte level was 29.6 x 10(9)/L (range 11.8 to 79.8), which was a 10-fold increase over baseline. The mean number of granulocytes collected was 41.6 x 10(9) (range 1.3 to 144.1), which was a six-fold increase over historical donors not receiving rhG-CSF. The mean granulocyte level 24 hours after transfusion into neutropenic recipients was 0.95 x 10(9)/L (median 0.57 and range .06 to 9.47). This study indicates that rhG-CSF is safe to administer to normal individuals, significantly improves the quantity of granulocytes collected, and results in significant circulating levels of granulocytes in neutropenic recipients. Further studies to evaluate rhG- CSF in normal granulocyte donors are warranted.     

No advantage for patients who receive splenic irradiation before bone marrow transplantation for chronic myeloid leukaemia: results of a prospective randomized study.

A total of 239 patients with chronic myeloid leukaemia (CML) in chronic phase awaiting bone marrow transplantation (BMT) from an HLA-identical sibling donor were randomized to receive, as part of their conditioning, splenic irradiation (SI+) or no splenic irradiation (SI-). There was no difference between the SI+ and SI- groups regarding the distribution of age, sex, donor/recipient sex combination and blood counts at diagnosis and at BMT. Survival, leukaemia-free survival (LFS), incidence of transplant-related mortality, incidence of rejection and probability of relapse do not differ between the 117 SI+ and the 118 SI- patients at a median follow-up time of 2.5 years (minimum 0.5 years). LFS at 30 months is 56% (SE 5%) for the SI+ and 51% (SE 6%) for the SI- group (p = 0.65). LFS is better for younger patients (less than 25 years), for patients without T cell depletion and for those with a low white blood cell count at diagnosis (less than 30 x 10(9)/l) (p less than 0.05). It is worst for male recipients of a female marrow (p less than 0.05). The incidence of graft-versus-host disease grade greater than or equal to II was higher in the SI+ group, though not significantly. We conclude that routine splenic irradiation prior to BMT for patients with CML is of no benefit and should not be used as a routine procedure.     

Transplantation of embryonic haemopoietic stem cells without prior recipient X-irradiation

Allogeneic day 7 mouse embryonic cells can colonize the haemopoietic system of normal, non-irradiated recipient mice. Donor embryonic cells are disaggregated and injected intravenously resulting in colonization in 40% of recipients, as shown by the presence of electrophoretic markers, characteristic of the donor cells. Donor type haemoglobin (Hb) and donor type glucose phosphate isomerase (GPI) demonstrates the presence of donor type erythrocytes and lymphocytes respectively. Repeat grafts in recipients not showing donor makers did not result in colonization. Recipient type haemopoiesis was dominant in all types of recipient. Skin grafts of allogenic donor type skin onto successfully grafted embryonic cell recipients did not survive. Allogeneic donor embryonic cells therefore survive in recipients where adult skin allografts do not. Donor embryonic cells, homozygous for T6 marker chromosomes, were used to assess the site of colonization of intravenously grafted cells. Donor chromosomes were seen in recipient liver and bone marrow at low levels. This distribution of donor cells persists for up to 64 d post-graft.     

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.     

Culture of hematopoietic stem cells purified from murine bone marrow.

The results of the Y-chromosome in situ hybridization experiments, the MRA assessment, and the long-term production of CFU-GM in vitro indicate that our protocol to sort low density WGA+, 15/1.1-, Rh123 dull cells enriches about 200-fold for PHSC. Assays for spleen colony formation (CFU-S) and radioprotection (30-day survival) were shown to be unspecific for PHSC, and, therefore, we lack a quantitative PHSC assay. The absolute number of PHSC in the bone marrow is not known any more, the purity of our sorted population likewise is unknown. Long-term repopulating cells (PHSC) could be separated from short-term repopulating ones by using Rh123 staining. The short-term repopulating cells (Rh123 bright) provided sufficient offspring to protect lethally irradiated mice until endogenous PHSC could reconstitute hematopoiesis. These cells are therefore of interest for bone marrow transplantation, because they provide radioprotection without long-term repopulation and graft-versus-host disease. For gene therapy these cells are of limited use, and PHSC with extensive replication are needed. The PHSC were not cultured successfully. Less than 15% of the sorted Rh123 dull cells responded in semisolid or liquid cultures in the presence of growth factors. Proliferation without differentiation was not observed. This may indicate that the right growth factor has not been found yet. On the other hand, about 30% of the cells responded in stromal layers of long-term bone marrow cultures and prolonged CFU-GM production and cobblestone area formation were observed there, suggesting that cell-cell contact and adherence molecules play a regulatory role in PHSC replication.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo administration of stem cell factor to mice increases the absolute number of pluripotent hematopoietic stem cells

We have examined the effects of administration of stem cell-factor (SCF) on the number and distribution of pluripotent hematopoietic stem cells (PHSC) in normal mice. Using the competitive repopulation assay we found that in vivo administration of SCF increases the absolute number of PHSC per mouse threefold. The increased numbers of PHSC are found in the peripheral blood and spleen of the SCF-treated animals. The spleen and peripheral blood stem cells completely repopulated the erythroid, myeloid, and lymphoid lineages of irradiated or W/Wv hosts, similar to bone marrow PHSC. PHSC from the peripheral blood of SCF- treated mice have a lineage marker-negative, c-kit-positive phenotype that is indistinguishable from that of bone marrow PHSC. The increase in the absolute number of spleen PHSC is associated with efficient gene transfer to these cells without prior treatment with 5-fluorouracil. This is a US government work. There are no restrictions on its use.     

Indomethacin augments granulocyte-macrophage colony-stimulating factor-induced hematopoiesis following 5-FU treatment

We have previously reported that granulocyte-macrophage colony-stimulating factor (GM-CSF) given after the administration of 5-fluorouracil (5-FU) results in augmented hematopoietic recovery as evidenced by increased white blood cell and neutrophil counts. Mice receiving GM-CSF following 5-FU administration were observed to have a marked elevation in splenic granulocyte-macrophage colony-forming cells (GM-CFC) and a decrease in the femoral bone marrow GM-CFC. Because GM-CSF has been shown to increase prostaglandin synthesis and prostaglandins are thought to provide a negative feedback signal to down-regulate myelopoiesis, we sought to determine if the cyclooxygenase inhibitor, indomethacin, could prevent the reduction in the number of femoral bone marrow GM-CFC seen when GM-CSF was administered following 5-FU. Groups of mice received a single 60 mg/kg i.p. injection of 5-FU followed 24 h later by twice-daily injections of 1 micrograms GM-CSF and daily injections of 3, 5, or 6 mg/kg indomethacin; the hematopoietic assays were performed on day 7 following 5-FU. Compared to those animals that received GM-CSF alone following 5-FU, mice receiving 5 mg/kg indomethacin plus GM-CSF following 5-FU had increased numbers of GM-CFC in their bone marrow (3923 +/- 634 vs 971 +/- 138; p less than 0.001) as well as increased neutrophil counts (18,995 +/- 2872 vs 11,497 +/- 2476; p less than 0.01). Indomethacin alone was, in part, capable of facilitating hematopoietic recovery following 5-FU administration, but not to the extent seen when used in combination with GM-CSF. Prostaglandin inhibitors may have a role in combination with hematopoietic growth factors in accelerating hematopoietic recovery following cytoreductive chemotherapy.     

In vivo stimulation of granulopoiesis by recombinant human granulocyte colony-stimulating factor.

Osmotic pumps containing Escherichia coli-derived recombinant human granulocyte colony-stimulating factor (rhG-CSF) were attached to indwelling jugular vein catheters and implanted subcutaneously into Golden Syrian hamsters. Within 3 days, peripheral granulocyte counts had increased greater than 10-fold with a concomitant 4-fold increase in total leukocytes. Microscopic examination of Wright-Giemsa-stained blood smears from rhG-CSF hamsters showed that only the neutrophil subpopulation of granulocytes had increased. No significant changes in lymphocyte or monocyte counts were observed during the course of continuous rhG-CSF treatment. After subcutaneous injection at rhG-CSF doses of up to 10 micrograms X kg-1 X day-1 only granulocyte counts were affected. However, at higher dose levels, a transient thrombocytopenia was noted. Erythrocyte had lymphocyte/monocyte counts remained unaffected by rhG-CSF over the entire dose range (0.3-300 micrograms X kg-1 X day-1) studied. Total leukocyte counts increased 3-fold within 12 hr after a single s.c. injection of rhG-CSF. This early effect was associated with an increase in the total number of colony-forming cells and the percent of active cycling cells in the marrow. A sustained elevation of peripheral leukocyte and marrow progenitor counts was observed following seven daily s.c. injections of rhG-CSF. The ability of rhG-CSF to increase the production and release of granulocytes from the marrow may underlie the beneficial effect it produced on the restoration of peripheral leukocyte counts in hamsters made leukopenic by treatment with 5-fluorouracil.