Effects of recombinant human tumor necrosis factor (rhTNF) on normal human and mouse hemopoietic progenitor cells

We examined the effects of recombinant human tumor necrosis factor (rhTNF) on normal human and murine granulocyte-macrophage (CFU-gm) and erythroid (CFU-e, BFU-e) progenitor cells. We suppressed in vitro colony formation by human marrow CFU-gm, CFU-e and BFU-e or peripheral blood BFU-e by adding rhTNF to the culture in a dose-related manner. A half-maximal inhibition was observed with 1-10 ng/ml. Leukemic cell line K562 cells were found to be sensitive to rhTNF in the clonogenic colony assay. However, the clonal growth of murine marrow CFU-e and BFU-e colonies was less than 50% inhibited and CFU-gm growth was unaffected even at a concentration of 1,000 ng/ml. We observed slight to moderate inhibition after 24 h pulse exposure of both human and murine-committed progenitors to rhTNF prior to the culture. Intravenous injection of 1 mg/kg of rhTNF caused a marked decrease in marrow erythroid progenitors and consequently caused anemia in the mice. Our data indicate that rhTNF has a suppressive effect on normal human and murine hemopoietic colony formation in vitro and murine erythropoiesis in vivo.     

Stimulation of retroviral vector infection of murine hematopoietic progenitors

Conditioned media (CM) from a cloned murine marrow-derived stromal cell line, AC6.21 (ALC), was shown to stimulate retroviral vector infection of hematopoietic progenitors in culture. Inclusion of ALC CM during cocultivation of normal murine bone marrow (BM) with vector-producing fibroblasts improved infection efficiency of day 13 spleen colony-forming cells (CFU-s) from 63% (15 provirus-positive spleen colonies/24 total), without added growth factor, to 90% (36 provirus-positive colonies/40 total). In addition, stimulation of BM cells with ALC CM during cocultivation improved retroviral infection of stem cells capable of repopulating the hematopoietic system of irradiated recipient animals. Because ALC CM was found to have 50 to 100 U/ml of IL-6 activity, purified recombinant human IL-6 was tested for an effect in this system. Stimulation with IL-6 alone increased retroviral infection efficiency of CFU-s from 15% (17 colonies provirus-positive/111 total analyzed) without added growth factor to 66% (97 provirus-positive colonies/148 total analyzed). These experiments support and extend previous studies which have demonstrated the necessity for growth factor stimulation in optimizing retroviral vector transduction of hematopoietic precursors.     

The effect of carbamylcholine on CFU-s differentiation

The proportion of spleen colony-forming units (CFU-s) killed by hydroxyurea was greatly increased after bone marrow cells (BMCs) from LACA mice were exposed to carbamylcholine (Cach; 1 X 10(-13) to 1 X 10(-9) in vitro and there was a marked change in the proportion of spleen colony types. Following treatment with Cach, granulocytic and mixed erythroid-type colonies increased from 20 to 26.3% and 16.1 to 29.6% in 9-day colonies and from 8.3 to 28.2% and 21.7 to 39.4% in 13-day colonies, respectively. Single cell suspensions of spleen colonies were made for granulocyte-macrophage progenitor (CFU-gm) and late erythroid progenitor (CFU-e) assays. The number of CFU-gm from Cach-treated BMC was about twice that from control BMC for both day 9 and day 13 groups; the number of CFU-e decreased relatively. The results suggest that cholinergic receptors on CFU-s may increase the tendency to differentiate into the granulocytic/monocytic line.     

The influence of histamine at various concentrations on the cell cycle state of hematopoietic stem cells (CFU-s)

The influence of histamine at various concentrations on the cell cycle state of hematopoietic stem cells (CFU-s) was investigated. CFU-s were triggered from the G0 state into the S phase of the cell cycle by in vitro treatment of mouse bone marrow cells with high concentrations of histamine. This effect could be antagonized by a histamine H2 receptor blocking agent. When bone marrow cells were treated with a histamine H1 receptor antagonist prior to histamine treatment, low concentrations of histamine also triggered the entrance of CFU-s into the DNA synthetic phase. Our findings further suggest the existence of histamine H1 and H2 receptors on the surface of CFU-s cells and the antagonistic effect of these two histamine receptor subtypes on the cell cycle state of CFU-s. Our results also suggest that histamine may participate in regulating the proliferation of hematopoietic stem cells in vivo during immune or inflammatory responses.     

Mast cell growth factor (C-Kit Ligand) in combination with granulocyte-macrophage colony-stimulating factor and interleukin-3:in vivo hemopoietic effects in irradiated mice compared toin vitro effects

In the presence of hemopoietic cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), mast cell growth factor (MGF; also known as steel factor, stem cell factor, and c-kit ligand) has proven to be a potent hemopoietic regulatorin vitro. In these studies, we examined thein vivo effects of MGF in combination with GM-CSF or GM-CSF plus IL-3. Effects were based on the ability of these cytokines to stimulate recovery from radiation-induced hemopoietic aplasia. Female B6D2F1 mice were exposed to a sublethal 7.75-Gy dose of⁶⁰Co radiation followed by subcutaneous administration of either saline, recombinant murine (rm) MGF (100g/kg/day), rmGM-CSF (100g/kg/day), rmIL-3 (100g/kg/day), or combinations of these cytokines on days 1–17 postirradiation. Recoveries of bone marrow and splenic spleen colony-forming units (CFU-s), granulocyte macrophage colony-forming cells (GM-CFC), and peripheral white blood cells (WBC), red blood cells (RBC) and platelets (PLT) were determined on days 14 and 17 during the postirradiation recovery period. MGF administered in combination with GM-CSF or in combination with GM-CSF plus IL-3 either produced no greater response than GM-CSF alone or down-regulated the GM-CSF-induced recovery. These results sharply contrasted results ofin vitro studies evaluating the effects of these cytokines on induction of GM-CFC colony formation from bone marrow cells obtained from normal or irradiated B6D2F1 mice, in which MGF synergized with GM-CSF or GM-CSF plus IL-3 to increase both GM-CFC colony numbers and colony size. These studies demonstrate a dichotomy between MGF-induced effectsin vivo andin vitro and emphasize that caution should be taken in attempting to predict cytokine interactionsin vivo in hemopoietically injured animals based onin vitro cytokine effects.Abbreviations GM-CSF Granulocyte-Macrophage Colonly-Stimulating Factor - IL-3 Interleukin-3 - MGF Mast Cell Growth Factor - SCF Stem Cell Factor - rm Recombinant Murine - CFU-s Colony Forming Unit-Spleen - GM-CFC Granulocyte Macrophage Colony-Forming Cell - WBC White Blood Cells - RBC Red Blood Cells - PLT Platelets - SLF Steel Factor - G-CSF Granulocyte Colonly-Stimulating Factor - IL-1 Interleukin-1 - IL-6 Interleukin-6 - Epo Erythropoietin - CFC Colony-Forming Cell - Sl Steel - BFU-e Erythroid Burst Forming Units - s.c Subcutaneous - PEG Polyethyleneglycol - PIXY321 GM-CSF/IL-3 Fusion Protein     

Macrophage colony-stimulating factor enhances monocyte and macrophage antibody-dependent cell-mediated cytotoxicity

In vitro culture of either human peripheral blood monocytes or murine peritoneal macrophages for 72 hr in the presence of macrophage colony-stimulating factor (M-CSF) dramatically increased their subsequent ability to mediate antibody-dependent cellular cytotoxicity (ADCC). The M-CSF-treated cells were more effective in ADCC at lower effector to target cell ratios and in the presence of lower concentrations of tumor-specific monoclonal antibody than the untreated control cells. Two other hematopoietic cytokines, granulocyte-macrophage colony-stimulating factor and interleukin-3, reported to enhance other macrophage effector functions were ineffective in promoting the development of ADCC by cultured human monocytes. All three hematopoietic growth factors were capable of enhancing the ability of the cultured monocytes to secrete TNF alpha; however, TNF alpha is unlikely to be an important cytotoxic factor in ADCC because neutralizing antibodies against TNF alpha had no affect on ADCC in vitro. Further, much higher concentrations of M-CSF were required to augment monocyte TNF alpha release (20-100 ng/ml) than ADCC capacity (1-10 ng/ml). These results suggest that M-CSF administration might prove effective in increasing the tumoricidal activities of tumor-specific monoclonal antibodies by enhancing the capacity of monocytes and macrophages to mediate ADCC.     

Up-regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in murine peritoneal exudate macrophages by both GM-CSF and IL-3.

Murine peritoneal exudate macrophages (PEM) display multiple CSF receptors. In this study, the expression of granulocyte-macrophage (GM)-CSF receptors in PEM was studied. PEM displayed over 5000 single type, high affinity GM-CSF receptors/cell with a Kd = 38 to 42 pM and an apparent molecular mass of 86,000 Da. Treatment of PEM with low, but not high, concentrations of recombinant murine (rMu) GM-CSF continuously for 24 h resulted in a marked up-regulation of GM-CSF receptors in PEM. A similar up-regulation of GM-CSF receptors also was detected in PEM cultures treated with rMuIL-3 (1-100 ng/ml) for 24 h or longer, regardless the doses of rMuIL-3 added in this case. Scatchard analysis of equilibrium binding showed that the enhanced binding activities in both cases were due to an increase in total number of GM-CSF receptors rather than changes in receptor affinity. Contrariwise, treatment with recombinant human macrophage-CSF (greater than 100-1000 ng/ml) partially inhibited the expression of GM-CSF receptors in PEM. Removal of rMuGM-CSF from culture medium 24 h after treatment led to a further up-regulation of GM-CSF receptors over a 4 to 24-h period, depending on the doses of initial treatment. On the other hand, removal of rMuIL-3 from culture medium after prolonged treatment did not result in further increase in GM-CSF receptors. The protein synthesis inhibitor cycloheximide abrogated GM-CSF receptor up-regulation induced by both rMuIL-3 and rMuGM-CSF, whereas actinomycin D inhibited only the second (8-24 h) phase of GM-CSF receptor up-regulation induced by exposure to high concentrations rMuGM-CSF (10 ng/ml). These findings suggest that rMuGM-CSF and rMuIL-3 up-regulate GM-CSF receptors in PEM in part through similar or identical metabolic pathways and provide further evidence of a close linkage between IL-3 and GM-CSF receptors.     

Effect of L-phenylalanine methyl ester on the colony formation of hematopoietic progenitor cells from human bone marrow

We have previously shown that L-phenylalanine methyl ester (PME) is capable of removing monocytes and enhancing the growth of hematopoietic colonies from human peripheral blood (PB) mononuclear cells (MNC). In the present study, we further compared the effect of PME on the colony formation of bone marrow (BM) and PB. Low density (less than or equal to 1.077 g/ml) MNC were obtained by Ficoll-diatrizoate density gradient centrifugation. Granulocyte/macrophage colony-forming units (CFU-gm) and erythroid burst-forming units (BFU-e) were cultured in agarose with conditioned media (CM) and/or interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF) and granulocyte/macrophage-CSF (GM-CSF). Treatment of BM MNC with 5 mM PME for 15 min at room temperature yielded a nucleated cell recovery of 44.8 +/- 5.0% (mean +/- SE; N = 8). CFU-gm were enriched 2.7-fold (range 2.0 to 4.8). Using CM or CM supplemented with G-CSF or GM-CSF has minimal effect on the enrichment. Leukocyte differentials revealed that 94.3 +/- 3.05% of the monocytes, as well as 91.2 +/- 1.60% of the cells in the neutrophilic maturation series were removed by PME. Incubation for 40 min in PME abolished CFU-gm formation. BFU-e were not enriched by the PME treatment. In contrast, 40 min incubation of PB MNC produced higher enrichment of CFU-gm than that obtained from 15 min of treatment, although lower cell recovery was obtained with the longer treatment time. In conclusion, we have demonstrated that phagocytic cells can be removed from BM or PB MNC by PME treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of growth hormone-stimulated lipolysis by somatostatin, insulin, and insulin-like growth factors (somatomedins) in vitro

The effects of somatostatin, insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II)/MSA on growth hormone (GH) (1 microgram/ml)-induced lipolysis were examined employing chicken adipose tissue in vitro. Basal and GH-stimulated glycerol release were inhibited by somatostatin (1 ng/ml) and by IGF-II/MSA (10 and 100 ng/ml). Insulin and IGF-I (10 and 100 ng/ml) completely inhibited the lipolytic response to GH without affecting basal glycerol release. Insulin and IGF-I were equipotent in inhibiting GH-induced lipolysis while IGF-II is only 16% as potent as insulin.     

Complement sensitivity of erythroid and myeloid precursors in paroxysmal in nocturnal hemoglobinuria

To test the hypothesis that paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder, the growth of BFU-e and CFU-gm and the complement sensitivity of cultured cells from BFU-e and CFU-gm colonies, as well as of unipotential progenitor cells (CFU-gm and BFU-e), were examined in five PNH patients. BFU-e growth was reduced in the three patients examined, and poor CFU-gm growth was noted in three of the five patients. Compared to normals, BFU-e and CFU-gm colonies in all patients demonstrated an increased susceptibility to the lytic action of complement when the release of 59Fe and myeloperoxidase was measured as specific markers for monitoring membrane damage. Compared to the growth of normal bone marrow cells, CFU-gm growth was significantly inhibited by pretreatment of bone marrow mononuclear cells with monoclonal OKIal antibody and complement. These findings support the proposition that a membrane defect predisposing blood cells to complement-mediated lysis may occur at the level of unipotential progenitor cells.     

Phagocytosis and killing of Staphylococcus aureus by granulocytes in mice after granulocyte-macrophage colony stimulating factor (GM-CSF) injection

GM-CSF is a hematopoietic growth factor. In vitro it stimulates the proliferation of myeloid progenitors and formation of granulocyte and macrophage colonies. It was found that GM-CSF in vitro is also stimulated the function of mature granulocytes, but we have no information about such influence in vivo. The purpose of this investigation was the evaluation in vivo of the GM-CSF effect on phagocytosis, bactericidal activity, and lysosome enzyme activities in granulocytes. GM-CSF was injected into mice subcutaneously during 5 consecutive days in the dose of 1 microgram/kg/d. The examination of the percent of cell phagocytizing bacteria (Staphylococcus aureus), NBT test, bactericidal activity and activation of acid phosphatase, alkaline phosphatase, peroxidase and esterase was performed every day and an evident increase of the tested parameters was found. These results prove in vivo activation of granulocytes by GM-CSF.     

Activation of adenosine A(3) receptors potentiates stimulatory effects of IL-3, SCF, and GM-CSF on mouse granulocyte-macrophage hematopoietic progenitor cells

Adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) has been tested from the point of view of potentiating the effects of hematopoietic growth factors interleukin-3 (IL-3), stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) on the growth of hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) in suspension of normal mouse bone marrow cells in vitro. IB-MECA alone induced no GM-CFC growth. Significant elevation of numbers of GM-CFC evoked by the combinations of IB-MECA with IL-3, SCF, or GM-CSF as compared with these growth factors alone has been noted. Combination of IB-MECA with G-CSF did not induce significantly higher numbers of GM-CFC in comparison with G-CSF alone. Joint action of three drugs, namely of IB-MECA + IL-3 + GM-CSF, produced significantly higher numbers of GM-CFC in comparison with the combinations of IB-MECA + IL-3, IB-MECA + GM-CSF, or IL-3 + GM-CSF. These results give evidence of a significant role of selective activation of adenosine A(3) receptors in stimulation of the growth of granulocyte/ macrophage hematopoietic progenitor cells.     

Zidovudine (AZT) treatment suppresses granulocyte-monocyte colony stimulating factor receptor type alpha (GM-CSFR alpha) gene expression in murine bone marrow cells

In vitro exposure of murine bone marrow cells to increasing concentrations of zidovudine (AZT, 0.1-50 microM) had a concentration dependent suppressive effect on the growth of granulocyte-monocyte colony forming unit (CFU-GM) derived colonies. In our previous published study, the mechanism of AZT-induced suppression of erythroid colony forming unit (CFU-E) derived colonies was linked to a decrease in erythropoitin receptor (Epo-R) gene expression. In this study, we have observed that AZT exposure also induced a concentration dependent suppressive effect (35-90%) on GM-CSF receptor type alpha (GM-CSFR alpha) gene expression. The suppression of GM-CSFR alpha mRNA expression was specific, since AZT caused a much lower decrease (15-22%) on the IL-3 receptor type alpha (IL-3R alpha) message level, and had an insignificant effect on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and c-myc message levels. Erythropoietin (Epo) therapy has been used for reversal of AZT induced erythroid toxicity. Exposure to increasing concentrations (10-500 U/ml) of GM-CSF was unable to override the suppressive effect of AZT on CFU-GM derived colonies, however, treatment in combination with IL-3 (10-250 U/ml) ameliorated the suppressive effects of AZT on CFU-GM and on GM-CSFR alpha and IL-3R alpha gene expression. These findings suggest a mechanism via which AZT may suppress granulocyte-monocyte specific differentiation in murine bone marrow cells. These data also suggest that a combination of GM-CSF and IL-3 may be a superior therapeutic intervention for AZT-induced neutropenia.     

OKT3 monoclonal antibody induces production of colony-stimulating factor(s) for granulocytes and macrophages in cultures of human T lymphocytes and adherent cells

OKT3 monoclonal antibody (mab) recognizes a membrane antigen associated with the T cell antigen recognition receptor, and is known to be mitogenic and to induce lymphokine production. Our studies demonstrate the ability of OKT3 mab to induce from cultures of human T lymphocytes supplemented with adherent cells the production of colony-stimulating factor(s) for granulocytes and macrophages (GM-CSF) and interferon-gamma (IFN-gamma), an inhibitor of clonal growth of hematopoietic progenitor cells. As has been shown for the mitogenic and IFN-gamma-inducing activity of OKT3 mab, the induction of GM-CSF release in cultures of T cells is strictly dependent on the presence of adherent cells. However, the concentrations of OKT3 mab required for optimal GM-CSF production (50 ng/ml) were found to be 80-fold higher than those sufficient for maximal IFN-gamma production, proliferation, and interleukin 2 production. IFN-gamma activity induced by OKT3 mab partially inhibited colony and cluster formation from progenitor cells of granulocytes and macrophages in vitro. Therefore, neutralization of the IFN-gamma by monoclonal anti-human-IFN-gamma antibody before assay of conditioned medium in bone marrow cultures significantly enhanced the detection of GM-CSF. Kinetic studies demonstrated maximal cumulative GM-CSF production in response to optimal OKT3 mab concentrations on days 4 through 6 in cultures of T cells supplemented with 15% adherent cells. Highly enriched OKT4+ and OKT8+ T cell subsets co-cultured with adherent cells in the presence of OKT3 mab both produced GM-CSF and IFN-gamma and showed similar dose-response curves to OKT3 mab. The requirement for the presence of adherent cells could not be overcome by the addition of purified interleukin 1 or macrophage supernatants. Studies using irreversible inhibitors of DNA (mitomycin C) or protein biosynthesis (emetine-HCl) revealed the necessity of intact DNA synthesis and translation in mononuclear cells to produce GM-CSF in response to OKT3 mab. Loss of GM-CSF production was observed when either adherent cells or T lymphocytes were treated with emetine before co-culture with untreated cells of the other population in the presence of OKT3 mab. In contrast, mitomycin C reduced GM-CSF production significantly when T cells, but not adherent cells, were pretreated. These results suggest that T lymphocytes and adherent cells closely cooperate in the production of GM-CSF induced by OKT3 mab.     

Expression of follicle-stimulating hormone receptor (FSHR) in goat ovarian follicles and the impact of sequential culture medium on in vitro development of caprine preantral follicles

This study evaluated the expression of FSH receptors (FSHR) in the different stages of goat follicle development and investigated whether the addition of increasing concentrations of FSH throughout the culture period influences the survival, growth and antral formation of in vitro-cultured caprine preantral follicles. The expression of FSHR was analysed before and after culturing follicles using real-time RT-PCR. For the culture, preantral follicles (≥150 μm) were isolated from ovarian fragments and cultured for 18 days in α-MEM+ alone or associated with recombinant FSH (rFSH: 100 or 1000 ng/ml), or in α-MEM+ supplemented with increasing concentrations of FSH throughout culture periods as follows: (a) sequential medium 1: FSH 100 ng/ml (from day 0 to 6), FSH 500 ng/ml (from day 6 to 12) and FSH 1000 ng/ml (from day 12 to 18); and (b) sequential medium 2: FSH 500 ng/ml (from day 0 to 9) and 1000 ng/ml (from day 9 to 18). Follicle development was evaluated on the basis of antral cavity formation, follicular and oocyte growth, and cumulus-oocyte complex health. The expression of FSHR in isolated caprine follicles increased from the preantral to antral phase. Regarding the culture, after 18 days, sequential medium 1 promoted follicular survival, antrum formation and a reduction in oocyte extrusion. Both sequential media promoted a higher rate of meiotic resumption compared with the other treatments. In conclusion, the addition of increased concentrations of FSH (sequential medium) has a significant impact on the in vitro development of caprine preantral follicles.     

Efficacy of an osmotic pump delivered,GM-CSF-based tumor vaccine in the treatment of upper aerodigestive squamous cell carcinoma in rats

PURPOSE: Upper aerodigestive tract (UADT) cancer has not experienced significant overall survival improvement for over 20 years, and no successful treatments for systemic disease exist. Most patients with UADT cancer experience immune suppression, therefore immune restorative therapies may offer promise for these patients. We presently tested the efficacy of granulocyte macrophage-colony stimulating factor (GM-CSF) delivered via 28-day continuous infusion pump, in combination with irradiated tumor cells, in a flank model of UADT cancer. METHODS: Five groups of rats were inoculated with syngeneic mucosally derived squamous carcinoma cells (FAT-7). Osmotic minipumps were implanted in the contralateral flank to deliver GM-CSF at 0 (PBS), 0.1, 1, 10, or 100 ng/day (n = 6 per group) for 28 days; 10(6) irradiated FAT-7 cells (ITC) were injected at the site of the GM-CSF infusion on days 0, 3, 7, 14, and 21 immune infiltrates in tumors were analyzed. RESULTS: Rats that received 10 or 100 ng/day GM-CSF/ITC had a significantly slower tumor growth rate compared to those who received 0, 0.1, or 1 ng/day (ANOVA, P < 0.01). There were increased CD 4+, CD 8+, and CD 68+ cells in tumors of GM-CSF/ITC treated animals over controls. CONCLUSION: GM-CSF (10 or 100 ng/day) delivered locally via osmotic pump with ITC slows the growth rate of mucosally derived squamous cell carcinoma in rats while improving immune cell infiltrates. The efficacy of locally delivered GM-CSF immunotherapy in this model may be a first step toward this immunotherapy strategy for humans.     

Granulocyte-macrophage colony-stimulating factor promotes the proliferation of human alveolar macrophages in vitro

The effects of granulocyte-macrophage (GM-CSF) or macrophage-CSF on in vitro proliferation of human alveolar macrophages (AM) were evaluated. AM of healthy volunteers incubated with recombinant human GM-CSF revealed incorporation of [3H]thymidine in vitro. The maximum incorporation was observed at 20 U/ml of GM-CSF on day 3. The proportion of proliferating cells incubated with 20 U/ml of GM-CSF from day 3 to day 4 was 8 to 11% of the total, whereas 3 to 5% of cells proliferated without GM-CSF. The number of cell nuclei increased 1.30- to 1.68-fold in the initial 7 days during incubation with 20 U/ml of GM-CSF, whereas there was a 1.07- to 1.13-fold increase without GM-CSF. Conditioned media obtained by the incubation with human lung tissue exhibited similar effects as recombinant human GM-CSF on macrophages. The effects were completely abrogated by antibody against human GM-CSF. Immunohistochemically, GM-CSF was detected in lung cells including AM, alveolar epithelium, alveolar interstitial cells, and endothelial cells. In contrast, recombinant macrophage-CSF did not induce the proliferation of human AM, although it has been known to promote the proliferation of murine AM. These observations suggest that GM-CSF plays an important role among the regulatory factors that locally support the population of AM in human lungs.     

Stem and stromal cell reconstitution of lethally irradiated mice following transplantation of hematopoietic tissue from donors of various ages

If the limited life span of hematopoietic tissues in vitro is due to a finite proliferative capacity of individual stem cells, one might expect tissues of young donors to possess a greater proliferative capacity and to contain a larger population of primitive stem cells than those of older donors. To test this hypothesis, we used 12- and 8-day spleen colony formation (CFU-s) to assay more and less primitive stem cell subpopulations of three murine hematopoietic tissues: fetal liver (FL) and weanling (WBM) and adult (ABM) bone marrow. Subsequently, the same assays and a stromal cell assay were performed on the bone marrow from groups of lethally irradiated mice reconstituted with these tissues. Comparison of the CFU-s content of the donor tissues revealed that FL contained a significantly greater proportion of primitive stem cells as evidenced by a (Day 12):(Day 8) CFU-s ratio of 3.0 +/- 1.0 as compared to 0.9 +/- 0.1 for WBM and ABM. In addition, at 21 weeks post-transplantation the CFU-s/femur values of the FL reconstituted group were significantly greater than those of the ABM and WBM reconstituted groups. These results suggest that fetal hematopoietic tissue contains a greater proportion of primitive stem cells and has a greater proliferative potential than hematopoietic tissue from older donors. No differences were seen in stromal cell reconstitution of the three experimental groups. In all cases, assayable fibroblast colony forming cells (CFU-f) remained at 20-40% of control values, even at 21 weeks postreconstitution.     

Stimulation of macrophage tumoricidal activity by the growth and differentiation factor CSF-1

The effect of the macrophage growth and differentiation factor CSF-1 on the tumoricidal capacity of murine peritoneal exudate macrophages was investigated. Pretreatment of peptone-elicited macrophages 1 day with 300-1200 U/ml CSF-1 induced moderate killing and greatly stimulated lymphokine (LK)-induced killing of [3H]thymidine-labeled TU5 sarcoma cells to levels above that seen with fresh macrophages. Further addition of CSF-1 at Day 1 at the time of the tumor lysis assay promoted moderate increases in spontaneous and LK-induced activity. CSF-1 did not stimulate freshly harvested exudate macrophages to lyse TU5 targets in the presence or absence of lymphokine (LK) activators. Lipopolysaccharide (LPS) at 0.1-1000 ng/ml did not stimulate cytotoxicity, and the low endotoxin content and the use of polymyxin B and C3H/HeJ mice excluded a role for LPS in these experiments. Incubation of the macrophages with IFN and the myeloid growth factors IL-3 and GM-CSF did not stimulate tumoricidal activity. CSF-1 has been proposed as a therapeutic agent to restore myeloid cell numbers in induced (cancer chemotherapy, bone marrow transplantation, etc.) and natural aplastic anemias. These studies show that CSF-1 also may be useful in combination with LK activators to promote resistance to cancer in mature mononuclear cells. CSF-1 may have similar effects in LK-activated macrophages to enhance resistance to infectious diseases.