Light-density Bone Marrow Cells Research Articles

Squalene Selectively Protects Mouse Bone Marrow Progenitors Against Cisplatin and Carboplatin-Induced Cytotoxicity In Vivo Without Protecting Tumor Growth

Squalene, an isoprenoid antioxidant is a potential cytoprotective agent against chemotherapy-induced toxicity. We have previously published that squalene protects light-density bone marrow cells against cis-diamminedichloroplatinum( II) (cisplatin)-induced toxicity without protecting tumor cells in vitro. Here, we developed an in vivo mouse model of cisplatin and cis-diammine (cyclobutane-1,1-dicarboxylato) platinum(II) (carboplatin)-induced toxicity to further investigate squalene-mediated LD-BM cytoprotection including the molecular mechanism behind selective cytoprotection. We found that squalene significantly reduced the body weight loss of cisplatin and carboplatin-treated mice. Light-density bone marrow cells from squalene-treated mice exhibited improved formation of hematopoietic colonies (colony-forming unit-granulocyte macrophage). Furthermore, squalene also protected mesenchymal stem cell colonies (colony-forming unit-fibroblast) from cisplatin and carboplatin-induced toxicity. Squalene-induced protection was associated with decreased reactive oxygen species and increased levels of glutathione and glutathione peroxidase/glutathione-S-transferase. Importantly, squalene did not protect neuroblastoma, small cell carcinoma, or medulloblastoma xenografts against cisplatin-induced toxicity. These results suggest that squalene is a potential candidate for future development as a cytoprotective agent against chemotherapeutic toxicity.

The Rate of Marrow Recovery and Extent of Donor Engraftment Following Transplantation of Ex Vivo–Expanded Bone Marrow Cells Are Independently Influenced by the Cytokines Used for Expansion

Successful stem cell transplantation depends on cell dose, and this is particularly true for placental/cord blood transplantation in which it has been clearly shown that both the success of engraftment as well as the speed of white cell and platelet recovery are dependent on the nucleated cell dose in the graft. Thus, if stem cell numbers could be increased, the speed as well as the likelihood of engraftment might be improved. We studied the effect of two different cytokine combinations--kit ligand (KL), interleukin-3 (IL-3), and Flt-3 ligand supplemented with thrombopoietin and IL-11 (combination 1) or granulocyte/macrophage colony-stimulating factor (GM-CSF) and G-CSF (combination 2)--for their ability to affect speed and extent of engraftment using limited numbers (5 x 10(4)) of murine bone marrow (BM) light-density (LD) cells or their progeny expanded ex vivo in the presence one or the other cytokine combination for 6 days. With combination 1, we found that speed of platelet recovery was enhanced, but at the expense of white blood cell (WBC) recovery and percent donor engraftment. Furthermore, the cytokine combination that best maintained donor engraftment, combination 2, did so at the expense of platelet recovery. In no case was percent donor engraftment improved over 5 x 10(4) unmanipulated LD BM cells. These results are consistent with the interpretation that immediate recovery of blood cells of different lineages and longterm donor engraftment are separate functions that can be influenced by the choice of cytokines used during the ex vivo expansion process.

Bone Marrow Transplantation or G-CSF Treatment Can Accelerate Recovery of Injured Spinal Cord in Mice.

Mice, unlike rats and humans, have a self recovery mechanism of spinal cord injury. Whether the hematopoietic system is involved in this mechanism is under investigation. In this study we tested whether bone marrow cells transplanted or mobilized by a growth factor in mice with spinal cord injury, can accelerate the recovery. C57bl/6 female mice 10 to 12 weeks of age underwent spinal cord incision in an open operation. The injury was performed as a complete transection including the dura mater and the whole circumference of the cord at the T10-T11 intervertebral space with a micro scalpel (No 11). Group A mice received 200μg/kg/day G-CSF subcutaneously for 7 days, starting 24 hours after operation. Group B mice received 106 light density bone marrow cells from C576bl/6 donor mice intravenously 24 hours after operation. Control group mice received no treatment. Histological evaluation was performed at 48 hours, 1 week, 3 weeks and 5 weeks postoperatively. Paraffin embedded longitudinal samples of spinal cord were cut as serial sections. Spinal cord damage was estimated by measuring the maximum diameter of the area of axonal damage and disruption of astrocytic network using immunostaining for neurofilaments and GFAP. Antibodies against CD68 were applied to identify macrophage aggregations. All measurements were performed by morphometric photo analysis. The volume of fibroblastic infiltration was estimated using a grading system (0–7), based on Van Gieson stain for connective tissue. Functional deficits and recovery over time were evaluated by testing hind limb reflex and coordinated motor function (Kuhn and Wrathal functional tests, modified by Seki et al, 2002). All tests have been videotaped. Outcome scores at 48 hours, 1 week, 3 weeks and 5 weeks postoperatively for the control group, group A and group B mice were analyzed with the Mann-Whitney U test. 48 hours post operatively all mice in all groups were paralyzed in both hind limbs. Gradual improvement was observed in all groups. At week 3 there was a significant difference between the mean scores of functional tests for both treated groups (A and B) compared with the mean scores of the control group. Statistically significant difference (p<0,05) was observed in 5 out of 7 tests for group A and in 3 out of 7 tests for group B. Same difference between Group A mice and control group mice was observed by 5 weeks, while group B had no statistically significant difference. No animal in any of the groups had a complete recovery 5 weeks postoperatively. Spinal cord in control group mice showed a gradually increase of fibroblastic infiltration until 5 week which entirely separated the two ends of the cord. In group A and group B mice a significant decrease of fibroblastic infiltration was observed at week 5 compared with week 3. Macrophage aggregations were evident at weeks 1 and 5 but not at week 3 in all groups. In conclusion our results indicate that light density bone marrow transplanted cells or G-CSF treatment can accelerate spinal cord injured mice recovery. It is possible that this is associated with a decrease in fibroblastic infiltration of spinal cord. Macrophage aggregation may also play an important role in the mechanism of recovery in mice, while in rats a different reaction including cavitation and delayed demyelination prohibits neurological recovery.

Haploidentical donor T cells fail to facilitate engraftment but lessen the immune response of host T cells in murine fetal transplantation.

The effects of donor T cells, or their CD8+ subset, on engraftment and tolerance induction in fetal transplantation were evaluated using an F1-into-parent mouse-model that does not permit a graft-versus-host effect. Gestational day 13 C57BL/6 (H-2Kb) fetuses were transplanted with B6D2F1 (H-2Kb/d) light density bone marrow cells (LDBMC) containing 1-2% T cells, T-cell depleted bone marrow cells (TDBMC, < 0.1% T cells), or TDBMC with enriched CD8+ T cells (CD8). Chimaerism levels in the peripheral blood, spleen and bone marrow were usually below 0.2% in all groups, indicating that T cells do not improve engraftment without a graft-versus-host effect. A significant, but transient, wave of donor cells was seen in the peripheral blood at 1 month of age in the CD8 and LDBMC groups. Relatively high levels of chimaerism (< 17%) were sometimes detected in the peritoneal cavities of recipients. T-cell tolerance specific to donor cells was evaluated in mixed lymphocyte cultures. The CD8 and LDBMC groups had significantly lower T-cell responses than untransplanted controls. These findings indicate that in utero transplantation of haploidentical donor CD8+ or CD3+ cells can help to lessen the immune response of host T cells towards donor cells. The persistence of donor cells in the peritoneal cavity also correlated with tolerance induction.

Murine bone marrow cells cultured ex vivo in the presence of multiple cytokine combinations lose radioprotective and long-term engraftment potential.

The desire to improve engraftment following transplantation of limited numbers of hematopoietic stem cells (HSC) has spurred the investigation of ex vivo stem cell expansion techniques. While surrogate outcomes, such as an increase in SCID-repopulating cells, suggest successful stem cell expansion in some studies, it is not clear that such assays predict outcomes using a more clinically relevant approach (e.g., myeloablation). We have addressed this by testing three cytokine combinations for their ability to increase the radioprotective and long-term marrow reconstitution capacity of hematopoietic cells cultured ex vivo. Low numbers of light-density (LD) mouse bone marrow (BM) cells or their expanded product were injected into lethally irradiated (9 Gy) congenic recipients. Survival rates and percent donor engraftment were compared at 2, 5, and 7 months post-transplant. The three cytokine combinations used were: (i) kit-ligand (L), thrombopoietin (Tpo), Flt-3 L; (ii) cytokines in (i) plus interleukin-11 (IL-11); (iii) cytokines in (ii) plus IL-3. At 7 months post-transplant, LD cell doses of 10(4), 2-2.5 x 10(4), and 0.5-1.0 x 10(5) gave predictable survivals of 20-30%, 40-70%, and 100%, respectively. Mean percent donor engraftments were 54.9% (SEM 36%), 55.7% (SEM 36%), and 76.3% (SEM 21%), respectively. When cells expanded for 3 or 5-7 days with the various cytokine combinations were transplanted into different groups of mice, survival rates and percent donor engraftment were almost uniformly poorer than results obtained with unmanipulated cells, and cells expanded for 5-7 days led to poorer outcomes than cells expanded for 3 days. Overall, ex vivo expansion of LD BM cells with the cytokine combinations chosen failed to improve transplant outcomes in this model.

Requirement for early donor cell chimerism during prolonged survival of murine skin allografts.

Posttransplantation infusion of donor bone marrow cells (BMC) can prolong allograft survival in antilymphocyte antibody- (ALA) treated recipients. This study examined the hypothesis that chimerism of donor BMC origin contributes to allograft unresponsiveness. Survival of day 0 skin grafts from C3H (H2k) donors was prolonged on ALA-treated B6AF1 mice by day +7 infusion of BMC from C3H or C3H-H2o2 (H2K(d)I(d)D(k)) mice. To test for functional chimerism, depletional anti-H2Kd antibody was injected at intervals after C3H-H2o2 BMC infusion. To confirm the persistence of active cells in the recipients, cells harvested from bone marrow of ALA- and C3H BMC-treated primary recipients were transferred to secondary ALA-treated recipients. Other recipients were infused on day +21 with additional donor BMC or light-density BMC that had been cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to promote the differentiation of dendritic cells (DC). Injection of depletional antibody targeting infused BMC interrupted skin graft survival, whether the injection was made on day +7, 2 hr after BMC injection, or as late as day +28. Prolonged graft survival was transferred to secondary recipients with cells recovered from primary recipient marrow as long as 4 weeks after initial donor BMC treatment. Graft survival prolonged by day +7 BMC was further enhanced by infusion on day +21 of light density cultured BMC, although a second dose of unfractionated BMC was inactive. Repeated injection of cultured BMC at 2-week intervals after day +21 prolonged graft survival even further. These data directly demonstrate that at least short-term donor BMC-derived microchimerism is required for prolonged allograft survival. The data further suggest that the active chimeric cells persist in recipient marrow. Finally, the beneficial effect of late infusion of cultured cells containing partially differentiated DC suggest that DC may be the active chimeric cells.

Defective Proliferation of Primitive Myeloid Progenitor Cells in Patients With Severe Congenital Neutropenia

AbstractAlthough several mechanisms have been proposed to explain the pathophysiology of severe congenital neutropenia (SCN), the precise defect responsible for SCN remains unknown. We studied the responsiveness of primitive myeloid progenitor cells to hematopoietic factors in 4 patients with SCN. The number of granulocyte-macrophage (GM) colonies formed in patients was decreased in response to granulocyte colony-stimulating factor (G-CSF) in both serum-supplemented and serum-deprived culture. The polymerase chain reaction–single-strand conformational polymorphism analysis of the G-CSF receptor gene showed no variance in structure conformation between the 4 patients and the normal subjects. In patients with SCN, the nonadherent light density bone marrow cells and cells that were purified on the basis of the expression of CD34 and Kit receptor (CD34+/Kit+ cells) showed the reduced response to the combination of steel factor (SF), the ligand for flk2/flt3 (FL), and interleukin-3 (IL-3) with or without G-CSF in serum-deprived culture. Furthermore, when individual CD34+/Kit+ cells from patients were cultured in the presence of SF, FL, and IL-3, with or without G-CSF for 10 days, the number of clones proliferated and the number of cells per each proliferating clone was significantly less than those in normal subjects. These results suggest that primitive myeloid progenitor cells of patients with SCN have defective responsiveness to not only G-CSF, but also the early- or intermediate-acting hematopoietic factors, SF, FL, and IL-3.

Defective Proliferation of Primitive Myeloid Progenitor Cells in Patients With Severe Congenital Neutropenia

Although several mechanisms have been proposed to explain the pathophysiology of severe congenital neutropenia (SCN), the precise defect responsible for SCN remains unknown. We studied the responsiveness of primitive myeloid progenitor cells to hematopoietic factors in 4 patients with SCN. The number of granulocyte-macrophage (GM) colonies formed in patients was decreased in response to granulocyte colony-stimulating factor (G-CSF) in both serum-supplemented and serum-deprived culture. The polymerase chain reaction-single-strand conformational polymorphism analysis of the G-CSF receptor gene showed no variance in structure conformation between the 4 patients and the normal subjects. In patients with SCN, the nonadherent light density bone marrow cells and cells that were purified on the basis of the expression of CD34 and Kit receptor (CD34(+)/Kit(+) cells) showed the reduced response to the combination of steel factor (SF), the ligand for flk2/flt3 (FL), and interleukin-3 (IL-3) with or without G-CSF in serum-deprived culture. Furthermore, when individual CD34(+)/Kit(+) cells from patients were cultured in the presence of SF, FL, and IL-3, with or without G-CSF for 10 days, the number of clones proliferated and the number of cells per each proliferating clone was significantly less than those in normal subjects. These results suggest that primitive myeloid progenitor cells of patients with SCN have defective responsiveness to not only G-CSF, but also the early- or intermediate-acting hematopoietic factors, SF, FL, and IL-3.

Serum granulocyte colony-stimulating factor levels in patients with chronic neutropenia of childhood: modulation of G-CSF levels by myeloid precursor cell mass

The serum G-CSF levels of eight patients with severe congenital neutropenia (SCN) were found to be significantly higher than those of 22 patients with chronic benign neutropenia (CBN). The relative number of cells expressing the G-CSF receptor in light density bone marrow cells (LDBMC) was lower in patients with SCN than in patients with CBN or in normal subjects. When recombinant human G-CSF was incubated with LDBMC, G-CSF levels were decreased by LDBMC from normal subjects and CBN patients, but not by those from SCN patients. Serum G-CSF concentrations, which are affected by mature neutrophils, may also be modulated by myeloid precursor cells in the bone marrow.

Serum granulocyte colony‐stimulating factor levels in patients with chronic neutropenia of childhood: modulation of G‐CSF levels by myeloid precursor cell mass

The serum G-CSF levels of eight patients with severe congenital neutropenia (SCN) were found to be significantly higher than those of 22 patients with chronic benign neutropenia (CBN). The relative number of cells expressing the G-CSF receptor in light density bone marrow cells (LDBMC) was lower in patients with SCN than in patients with CBN or in normal subjects. When recombinant human G-CSF was incubated with LDBMC, G-CSF levels were decreased by LDBMC from normal subjects and CBN patients, but not by those from SCN patients. Serum G-CSF concentrations, which are affected by mature neutrophils, may also be modulated by myeloid precursor cells in the bone marrow.

Effect of nitric oxide production and oxygen tension on progenitor preservation in ex vivo culture

Many cell types are capable of expressing inducible nitric oxide synthase (iNOS) in response to cytokines or endotoxin. We detected iNOS mRNA by reverse transcriptase polymerase chain reaction in CD34 + human bone marrow cells after 48-hour incubation with interferon-gamma (IFN-γ)/tumor necrosis factor alpha (TNF-α) and IFN-γ/ lipopolysaccharide. Based on this finding, we examined the effect of nitric oxide (NO) on hematopoiesis and particularly on proliferation and survival of CD34 + marrow cells in in vitro culture. When CD34 + cells were cultured in the presence of an NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a dose-dependent inhibition of cell proliferation was observed. Addition of the selective iNOS inhibitor, L-N 6-(1-iminoethyl)-lysine hydrochloride (L-NIL) at a dose of 500 μM increased the cell counts by 23% (range 0–89%). The expansion of total CD34 + cell number was 4-fold with a hematopoietic cytokine cocktail compared to 5.2-fold with the addition of L-NIL to this combination. At days 7 and 14 of culture, SNAP induced apoptosis in CD34 + human bone marrow cells detected by an in situ terminal deoxynucleotidyl transferase assay. The apoptosis was partially inhibited with the addition of L-NIL. SNAP also inhibited cell cycling, as evidenced by a 56% decrease in the number of cells in S phase after 5 days of culture in the presence of SNAP. To investigate if NO production was dependent on oxygen tension, J774A mouse macrophage cells were induced with lipopolysaccharide/IFN-γ, and nitrite production measured by the Griess reaction. Nitrite production was persistently less in 5% compared to 21% oxygen. CD34 + marrow cells from normal donors were also grown under variable oxygen tension, and the cell proliferation in 5% oxygen was significantly greater at both 7 and 14 days of culture. CFU-GM colony growth also was increased in the low oxygen setting. The concentration of varous cytokines was measured in CD34 + progenitor culture supernatants, including interleukin (IL)-1α, IL-1β and TNF-α. SNAP increased IL-1α production by CD34 + cells as well as from light-density bone marrow cells, whereas the effect on IL-1β and TNF-α production was not significant. Manipulation of oxygen tension and the inhibition of production of reactive oxygen and nitrogen intermediates may have potential to optimize cell expansion and progenitor preservation in ex vivo culture systems.

Constitutive expression of p53 protein in enriched normal human marrow blast cell populations [see comments

Previous studies by others using metabolic labeling, cell lysis, and immunoprecipitation have reported elevated levels of p53 protein in blast cells derived from patients with acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML), whereas p53 protein was not detected in normal light-density bone marrow cells. In this report, using the same detection methods, we confirm the negligible expression of p53 protein in normal light density marrow cells. However, we find clearly significant levels of p53 protein expression in enriched normal human marrow blast populations. Furthermore, using a panel of p53 specific monoclonal antibodies, we find the p53 protein constitutively synthesized by normal marrow blasts has the immunologic phenotype identified by PAb240 that reportedly recognizes a common conformational-dependent epitope on mutant p53. We have also found that the p53 immunologic subclass identified by PAb240 exists in normal human circulating lymphocytes either resting, serum starved, or PHA activated. In summary, it is clear that (1) normal marrow blast populations provide the appropriate control for assessing the levels of p53 protein expression in leukemic blast cells; and (2) PAb240 cannot be used to distinguish p53 mutated at the DNA level from normal p53 in fresh human hematopoietic cells.

Constitutive Expression of p53 Protein in Enriched Normal Human Marrow Blast Cell Populations

Abstract Previous studies by others using metabolic labeling, cell lysis, and immunoprecipitation have reported elevated levels of p53 protein in blast cells derived from patients with acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML), whereas p53 protein was not detected in normal light-density bone marrow cells. In this report, using the same detection methods, we confirm the negligible expression of p53 protein in normal light density marrow cells. However, we find clearly significant levels of p53 protein expression in enriched normal human marrow blast populations. Furthermore, using a panel of p53 specific monoclonal antibodies, we find the p53 protein constitutively synthesized by normal marrow blasts has the immunologic phenotype identified by PAb240 that reportedly recognizes a common conformational- dependent epitope on mutant p53. We have also found that the p53 immunologic subclass identified by PAb240 exists in normal human circulating lymphocytes either resting, serum starved, or PHA activated. In summary, it is clear that (1) normal marrow blast populations provide the appropriate control for assessing the levels of p53 protein expression in leukemic blast cells; and (2) PAb240 cannot be used to distinguish p53 mutated at the DNA level from normal p53 in fresh human hematopoietic cells.

In vivo interleukin-1 (IL-1) administration indirectly promotes type II IL-1 receptor expression on hematopoietic bone marrow cells: novel mechanism for the hematopoietic effects of IL-1

Interleukin-1 (IL-1) has profound stimulatory effects on hematopoiesis but the mechanism(s) of action remain unknown. The direct action of IL- 1 on hematopoietic progenitor cells requires the presence of a specific IL-1 receptor (IL-1R). In this report, we tested the effect of in vivo IL-1 treatment on the expression of IL-1R on bone marrow (BM) cells. Injection of mice with IL-1 results in a marked upregulation of IL-1R on light-density BM cells as on a subpopulation enriched for myeloid precursors. Pretreatment of mice with anti-type I IL-1R antibody (35F5), which has been shown to prevent the radioprotective effect of IL-1, also blocked IL-1-induced IL-1R expression on BM cells. This antibody did not directly bind and block IL-1 binding to the type II IL- 1R expressed on hematopoietic cells, suggesting that IL-1R upregulation by IL-1 is indirect. It is therefore possible that IL-1 acts on type I IL-1R-expressing accessory cells such as stromal cells or T cells to induce production of hematopoietic growth factors (HGFs). In support of this, granulocyte colony-stimulating factor administration can induce the increase of IL-1R on BM cells. Thus, the increased expression of IL- 1R on hematopoietic BM cells by IL-1 is indirect, probably mediated in part through endogenous HGF production. These results also suggest that the restorative hematopoietic effect of IL-1 occurs through both indirect and direct mechanisms.

In Vivo Interleukin-1 (IL-1) Administration Indirectly Promotes Type II IL-1 Receptor Expression on Hematopoietic Bone Marrow Cells: Novel Mechanism for the Hematopoietic Effects of IL-1

Interleukin-1 (IL-1) has profound stimulatory effects on hematopoiesis but the mechanism(s) of action remain unknown. The direct action of IL-1 on hematopoietic progenitor cells requires the presence of a specific IL-1 receptor (IL-1R). In this report, we tested the effect of in vivo IL-1 treatment on the expression of IL-1R on bone marrow (BM) cells. Injection of mice with IL-1 results in a marked upregulation of IL-1R on light-density BM cells as on a subpopulation enriched for myeloid precursors. Pretreatment of mice with anti-type I IL-1R antibody (35F5), which has been shown to prevent the radioprotective effect of IL-1, also blocked IL-1-induced IL-1R expression on BM cells. This antibody did not directly bind and block IL-1 binding to the type II IL-1R expressed on hematopoietic cells, suggesting that IL-1R upregulation by IL-1 is indirect. It is therefore possible that IL-1 acts on type I IL-1R-expressing accessory cells such as stromal cells or T cells to induce production of hematopoietic growth factors (HGFs). In support of this, granulocyte colony-stimulating factor administration can induce the increase of IL-1R on BM cells. Thus, the increased expression of IL-1R on hematopoietic BM cells by IL-1 is indirect, probably mediated in part through endogenous HGF production. These results also suggest that the restorative hematopoietic effect of IL-1 occurs through both indirect and direct mechanisms.

Inhibition of human macrophage colony formation by interleukin 4.

We investigated the effects of human rIL-4 on in vitro hematopoiesis. A profound inhibition of macrophage colony formation by IL-4 was observed, whereas colony growth of other lineages was not affected. Inhibition of macrophage colony growth was not restricted to GM-CSF-induced colony growth but was also present in cultures stimulated with M-CSF. This inhibition was not only observed in cultures of light density bone marrow cells, but also in cultures of monocyte- and T lymphocyte-depleted bone marrow cells. Since a similar inhibition was observed in cultures of CD34+HLA-DR+-enriched bone marrow cells, a direct action of IL-4 on monocyte-committed progenitor cells is suggested.

Biosynthetic (Recombinant) Human Granulocyte-Macrophage Colony-Stimulating Factor: Effect on Normal Bone Marrow and Leukemia Cell Lines

To examine the biologic properties of the molecule encoded by the human gene for granulocyte-macrophage colony-stimulating factor (GM-CSF), we expressed the cloned complementary DNA (cDNA) in transfected monkey COS cells and purified the resultant protein. Purified biosynthetic human GM-CSF was added to cultures of normal hematopoietic progenitor cells in semisolid media, and the resulting colonies were characterized cytochemically. Non-adherent light-density bone marrow cells from healthy adult volunteers were maximally stimulated with GM-CSF (approximately 250 pmol/L, and four types of colonies were consistently identified by aspirating the individual colonies and staining with a triple stain for specific and nonspecific esterases and eosinophilic granules. Pure neutrophilic granulocyte (G), mixed granulocyte-macrophage (GM), pure macrophage (M), and pure eosinophil (EO) colonies were observed, the mean incidences on day 8 being 70%, 20%, 5%, and 5%, and on day 14, 7.5%, 16.6%, 50.9%, and 25.0%, respectively. In all types of colonies, complete maturation to segmented forms or typical macrophages was detected. GM-CSF did not enhance the growth of BFU-E from normal peripheral blood buffy coat cells in the simultaneous presence of erythropoietin alone or erythropoietin with purified erythroid-potentiating activity. GM-CSF stimulated HL-60 and KG-1 colony formation twofold and fivefold, respectively; consistent differentiation induction towards monocytic and eosinophilic lineages was observed in HL-60 but not in KG-1. These in vitro findings indicate that GM-CSF is a multilineage stimulator for progenitor cells of G, GM, M, and EO colonies.

Biosynthetic (recombinant) human granulocyte-macrophage colony- stimulating factor: effect on normal bone marrow and leukemia cell lines

To examine the biologic properties of the molecule encoded by the human gene for granulocyte-macrophage colony-stimulating factor (GM-CSF), we expressed the cloned complementary DNA (cDNA) in transfected monkey COS cells and purified the resultant protein. Purified biosynthetic human GM-CSF was added to cultures of normal hematopoietic progenitor cells in semisolid media, and the resulting colonies were characterized cytochemically. Non-adherent light-density bone marrow cells from healthy adult volunteers were maximally stimulated with GM-CSF (approximately 250 pmol/L, and four types of colonies were consistently identified by aspirating the individual colonies and staining with a triple stain for specific and nonspecific esterases and eosinophilic granules. Pure neutrophilic granulocyte (G), mixed granulocyte- macrophage (GM), pure macrophage (M), and pure eosinophil (EO) colonies were observed, the mean incidences on day 8 being 70%, 20%, 5%, and 5%, and on day 14, 7.5%, 16.6%, 50.9%, and 25.0%, respectively. In all types of colonies, complete maturation to segmented forms or typical macrophages was detected. GM-CSF did not enhance the growth of BFU-E from normal peripheral blood buffy coat cells in the simultaneous presence of erythropoietin alone or erythropoietin with purified erythroid-potentiating activity. GM-CSF stimulated HL-60 and KG-1 colony formation twofold and fivefold, respectively; consistent differentiation induction towards monocytic and eosinophilic lineages was observed in HL-60 but not in KG-1. These in vitro findings indicate that GM-CSF is a multilineage stimulator for progenitor cells of G, GM, M, and EO colonies.

Mechanism of Action of Serum Factors That Regulate Granulopoiesis In Vitro: Possible Physiologic Role of Serum-Inhibiting Activity

The purpose of this study was to investigate mechanisms of action and in vivo relationships of serum factors that regulate granulopoiesis in vitro. Granulopoiesis involves interactions between stimulating and proliferating cells, and in order to determine mechanisms of action it is necessary to assess whether an activity acts on the stimulating or on the proliferating cell. Liquid cultures of monocytes, obtained from light-density white blood cells, were used to observe effects on colony-stimulating factor (CSF) production, and soft agar cultures of nonadherent light-density bone marrow cells were used to determine effects on colony-forming cells. Medium molecular weight serum-stimulating activity increased monocyte CSF production but had no effect on colony-forming cells. High molecular weight serum-inhibiting activity had no effect on CSF production but inhibited the action of CSF. Serum-inhibiting activity correlated with the circulating neutrophil concentration. These observations suggest that serum stimulation of granulopoiesis is achieved indirectly by stimulating marrow CSF-producing cells and that growth of the proliferating cell population is stabilized by a direct negative feedback loop.

Nutritional and Regulatory Roles of Human Serum in Cultures of Human Granulopoietic Cells

We proposed to develop a tissue culture medium supplement based on human serum that would support proliferation of human marrow cells and then to use it in an examination of human serum for factors that regulate colony formation. We examined various serum treatments (heat, dialysis, ultrafiltration) and found that heat-treated human serum (58°C for 120 min) retained the ability to support proliferation and that this heat treatment eliminated the variability between different sera at concentrations > 30%. We concluded that tissue culture medium, supplemented with 30% heat-treated human serum, was adequate and that the use of this growth medium would complement an examination of human serum for thermolabile factors. To reduce cell-cell interactions in culture to a minimum, we used cell separation procedures and employed nonadherent light-density bone marrow cells (NALD BMC) for colony-forming cells, light-density white blood cells (LD WBC) for colony-stimulating cells, and LD WBC-conditioned medium for colony-stimulating factor. We examined human serum, whole and fractionated (Sephadex G200), for effects on colony formation in agar cultures supplemented with 30% heat-treated human serum containing NALD BMC only, NALD BMC plus LD WBC, and NALD BMC plus conditioned medium. We found that human serum contained thermolabile inhibitory and stimulatory activities that regulated colony formation in NALD BMC cultures stimulated with LD WBC. Neither whole nor fractionated human serum induced colony formation in NALD BMC cultures. This suggests that the colony-stimulating activity of human serum is not due to colony-stimulating factor.