Haemopoietic colony formation (BFU-E, GM-CFC) during the development of pure red cell hypoplasia induced in the cat by feline leukaemia virus

The GM-CFC assay for granulocyte-macrophage progenitors and the BFU-E and CFU-E assay for early and late erythroid progenitors from cat bone marrow were characterized. GM-CFC gave 59 +/- 4 to 118 +/- 6 colonies per 10(5) bone marrow cells using colony stimulating factors (CSF) from cat, mouse or human sources. The CFU-E and BFU-E assays gave 114 +/- 7 and 58 +/- 7 colonies respectively with optimum doses of erythropoietin. Irradiated cat bone marrow cells were good sources of CSF and of burst promoting activity for these assays. Kittens infected with feline leukaemia virus, subgroup C (FeLV-C), which induces pure red cell hypoplasia, showed the incidence of BFU-E decreased to 25-35% of controls as early as one week postinfection, and even lower values at later times. In contrast, the incidence of GM-CFC remained normal for several weeks. No evidence of inhibitory cells or of lack of stimulatory cells in the infected marrows was seen when they were cultured together with normal marrow in the BFU-E assay. Conversely, normal marrow cells were not able to restore BFU-E growth from infected marrow. This suggests a direct action of FeLV-C on early erythroid precursors. Infection with FeLV, subgroup A, which induces only a mild transitory anaemia, produces only a moderate decrease in the incidence of BFU-E.     

Demonstration of retroviral proteins associated with erythroid progenitors of cats with feline leukemia virus-induced erythroid aplasia

Feline erythroid aplasia is a fatal retrovirus-induced disease related to infection with feline leukemia virus of subgroup C. When bone marrow cells from cats inoculated with FeLV were incubated with polyvalent antibody to FeLV and cultured for erythroid colony formation, a complement-dependent inhibition of erythroid progenitors was demonstrated with virtually complete suppression of erythroid colony-forming units (CFU-E) and burst-forming units (BFU-E) evident from post-inoculation week 4 until termination in cats with progressive infection. When bone marrow cells from cats with regressive infection were treated similarly, CFU-E were inhibited on post-inoculation week 4 (98% inhibition) when the cats were viremic, and on week 6 (77% inhibition) when the leukocytes were negative for FeLV by immunofluorescence. These data suggest that the retroviral proteins are associated with erythroid progenitors or critical accessory cells and may play a role in selective suppression of erythropoiesis.     

Interaction between feline leukaemia virus subgroups in the pathogenesis of erythroid hypoplasia

The interaction of feline leukaemia virus (FeLV) of subgroups A and C in the pathogenesis of erythroid hypoplasia in cats was studied. Weanling kittens infected with FeLV-A became permanently viraemic but remained haematologically normal over a period of 36 weeks. Similar kittens inoculated with FeLV-C, which produces erythroid hypoplasia when administered to newborn kittens, neither became viraemic nor developed the disease. However, weanling kittens inoculated with a mixture of FeLV-A and C became viraemic, first with FeLV-A and then additionally with FeLV-C, and the emergence of FeLV-C into the blood coincided with the advent of erythroid hypoplasia. When FeLV-C was inoculated into five older cats which had been viraemic with FeLV-A for several months previously, it appeared in the plasma of three of the cats and erythroid hypoplasia was diagnosed in two of these, 16-20 weeks after infection with FeLV-C. These results show that FeLV-A enhances the growth of FeLV-C in cats and overcomes their age-related resistance to FeLV-C. Also, the appearance of FeLV-C in the plasma of cats viraemic with FeLV-A indicates that erythroid hypoplasia will subsequently occur rapidly. These findings are relevant to the origin of FeLV-C isolates and their occurrence in nature.     

Effect of tumor necrosis factor on human hematopoietic precursor cells

The effect of human recombinant tumor necrosis factor (rhTNF-alpha) on the in vitro colony growth of normal hematopoietic progenitor cells was investigated. In a clonal colony-assay a dose-dependent inhibition of erythroid BFU-E, granulocyte/monocyte CFU-GM and megakaryocyte CFU-Mk was demonstrated. CFU-Mk were completely inhibited by low doses of TNF-alpha (3 U-300 U/ml). 50% inhibition occurred at 10 U/ml of TNF for CFU-Mk, 100% inhibition at 300 U/ml of TNF. 50% inhibition occurred at 233 U/ml of TNF for CFU-GM, and 100% inhibition for CFU-GM was not observed. For inhibition of BFU-E higher doses of TNF-alpha were necessary (100 U-1,000 U/ml). The growth inhibitory effect could selectively be abolished by antibodies against TNF-alpha. Removal of adherent cells and T-lymphocytes from the bone marrow cells had no significant influence of the suppressive effect of TNF-alpha. The inhibitory effect of TNF-alpha is due to a direct action on hematopoietic progenitor cells and not mediated by accessory cells.     

The effects of transforming growth factor beta 3 on the growth of highly enriched hematopoietic progenitor cells derived from normal human bone marrow and peripheral blood

The effects of transforming growth factor beta 3 (TGF-beta 3) on growth in semisolid cultures of enriched hematopoietic progenitors derived from normal human marrow and blood were evaluated. Conditioned media from the Mo-T cell line (MoCM) were the source of colony-stimulating factors used to optimally stimulate primitive progenitors. To assess whether a proportion of granulocyte/monocyte (GM) progenitors were prevented from cycling, all sizes of GM aggregates were evaluated from 3 to 20 days. The activity of TGF-beta 3 on the growth of erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM) was similar to that observed for TGF-beta 1. TGF-beta 3 (10, 100, and 1,000 pmol/liter), added initially or 72 h after initiation of culture, did not significantly affect the total number of marrow GM aggregates at 3, 7, 14, and 20 days, but TGF-beta 3 (1,000 pmol/liter), added initially, reduced the total number of blood GM aggregates. This suggests that some blood GM progenitors might be blocked from cycling but that the great majority of marrow GM progenitors are not blocked. Whether TGF-beta 3 (10, 100, and 1,000 pmol/liter) was added initially or after 72 h of stimulation by MoCM, there was a dose-dependent reduction of marrow and blood GM colony size even when the total number of colonies was unaffected. TGF-beta 3 (10, 100, and 1,000 pmol/liter), added initially or at 72 h, reduced in a dose-dependent manner the size of marrow and blood-derived BFU-E. TGF-beta 3 (1,000 pmol/liter) was more likely to reduce the total number of marrow and blood BFU-E, and this increased sensitivity of the erythroid lineage may prevent the development of this population in colonies derived from multipotential colony-forming unit-granulocyte/erythroid/monocyte (CFU-GEM). The results suggest that the main effect of TGF-beta 3 and TGF-beta 1 is to slow the rate of proliferation of hematopoietic progenitors rather than to prevent them from beginning proliferation. This results in a reduction in colony size which prevents the identification of primitive versus mature progenitor on the basis of standard criteria of colony size.     

Selective depletion of human myeloma clonogenic stem cells from bone marrow cell preparations by a plasma-cell reactive antibody and complement

The monoclonal antibody MM4 reacts with human myeloma cells from plasma cell dyscrasia (PCD)-derived cell lines and bone marrow (BM) biopsies from PCD patients, but not with normal BM or peripheral blood mononuclear (PBM) cells. We examined cytotoxicity of MM4 and rabbit complement (MM4:C') on mixtures of normal BM mononuclear cells and myeloma cells from three different PCD-derived cell lines, RPMI 8226, GM 1312, or ARH-77. For cell preparations containing 10% myeloma cells, treatment with MM4 (500 micrograms per 10(5) cells, 4 degrees C, 60 min) and two cycles of complement (1:8, 23 degrees C, 2 x 30 min) consistently eliminated 2 logs or more of clonogenic myeloma stem cells, as determined by colony growth assays and limiting dilution analysis (99.4%, 98.9%, and 99.96% reduction of RPMI 8226, GM 1312, and ARH-77 cells, respectively). The majority of normal marrow progenitors were spared (inhibition of CFU-C: 10-13%; BFU-E: 0%). These observations suggest that MM4 may be useful for selective depletion of human myeloma clonogenic stem cells from bone marrow ex vivo.     

Enhancement of the proliferation of murine fetal liver erythroid progenitors by infection with Harvey sarcoma virus

We recently developed a new progenitor assay using murine fetal liver cells that provides a source of pluripotent progenitors, bipotent progenitors, and committed macrophage, megakaryocyte, erythroid, and mast cell progenitors. This clonal cell culture system was used to examine the direct effects of Harvey sarcoma virus on murine hemopoietic progenitors. Very large erythroid colonies containing 100,000 to 200,000 cells were seen in the infected group. Only small erythroid colonies were seen in the uninfected control cultures. The cells in the large erythroid colonies from infected cultures expressed the ras gene as demonstrated by immunofluorescence with a monoclonal antibody to p21, the ras gene product. The infected cells were not immortal since they did not yield secondary colonies upon replating. Sequential observation of individual colonies showed that maturation was not blocked by infection with the virus. The size of other colony types, including granulocyte/macrophage, mast cell, and mixed, was unaffected even though some of these colonies expressed the ras gene. Thus, infection with Harvey sarcoma virus appears to give a growth advantage primarily to committed erythroid progenitors.     

Effects of recombinant growth factors on radiation survival of human bone marrow progenitor cells

The purpose of this study was to evaluate the individual radioprotective effects of 4 distinct purified recombinant human hematopoietic growth factors, namely recombinant human granulocyte-macrophage colony stimulating factor (rGM-CSF), recombinant human granulocyte colony stimulating factor (rG-CSF), recombinant human interleukin 1 (rIL-1), and recombinant human interleukin 2 (rIL-2) on human myeloid (CFU-GM) and erythroid (BFU-E) bone marrow progenitor cells. We demonstrate that (a) preconditioning with rGM-CSF, rG-CSF, or rIL-1 enables CFU-GM to repair sublethal radiation damage and renders CFU-GM less radiosensitive, (b) preconditioning with rGM-CSF or rIL-1 enables BFU-E to repair sublethal radiation damage, and (c) preconditioning with rIL-2 does not increase the radiation survival of CFU-GM or BFU-E. The effects of recombinant growth factors, in particular rGM-CSF, on the radiation damage repair, radiosensitivity, and proliferative activity of bone marrow progenitor cells resulted in a substantial increase in the mean numbers of progenitor cell-derived hematopoietic colonies in irradiated marrow samples. The effects of rGM-CSF on the radiation response of CFU-GM and BFU-E, and the effects of rG-CSF as well as rIL-1 on the radiation response of CFU-GM did not appear to require the presence of T-cells/T-cell precursors, NK-cells, B-cells/B-cell precursors, monocytes, macrophages, MY8 antigen positive non-CFU-GM myeloblasts, promyelocytes, myelocytes, metamyelocytes, granulocytes, or glycophorin A positive erythroid cells since virtually identical results were obtained with unsorted marrow samples or highly purified fluorescence activated cell sorter (FACS) isolated progenitor cell suspensions. To our knowledge, this report represents the first study on recombinant human growth factor-induced modulation of the radiation responses of normal human bone marrow progenitor cells.     

In vitro studies of erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes

Erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes (MDS) was evaluated by measuring the in vitro response of primitive (BFU-E) and relatively mature (CFU-E) erythroid progenitors from 12 patients and from eight healthy donors to recombinant human erythropoietin (rhEPO), and by quantifying relationships between circulating EPO levels and progenitor cell frequencies in MDS marrow. Half-maximal growth of MDS CFU-E and BFU-E was detected at a 4-fold higher rhEPO concentration than required by control erythroid progenitors. Nine of the patients evaluated exhibited maximal growth of erythroid colonies at 5- to 20-fold higher than control saturating rhEPO concentrations. Circulating EPO levels in MDS patients were elevated, with a mean value approximately 35-fold higher than that of controls. The frequency of MDS marrow CFU-E and BFU-E was 57 +/- 42% and 18 +/- 9% of the mean control values, respectively. Correlation analysis of the relationships between MDS EPO levels and erythroid progenitors indicated that the anemia in MDS is not attributable to an abnormality in the capacity of EPO to induce the generation of CFU-E, but may be influenced by the BFU-E population, whose severe deficiency results in insufficient influx of EPO-responsive cells. Our findings therefore suggest that treatment of MDS patients with rhEPO may be of limited benefit, since the generation of BFU-E from more primitive ancestors and the initial growth requirements of these cells are not under the regulatory influence of this hormone.     

Effect of lymphokine-activated killer cell fraction on the development of human hematopoietic progenitor cells

Lymphokine-activated killer (LAK) cells from cultures of human peripheral blood mononuclear cells with recombinant interleukin-2 (rIL-2) have been clinically used in adoptive immunotherapy for cancer patients. To study their influence on human hematopoiesis, the LAK cell fraction was cocultured with marrow nonphagocytic cells from normal subjects in an assay system of hematopoietic progenitors. The fraction suppressed colony growth from relatively mature erythroid progenitors in a dose-dependent manner. Although unactivated cells, which were produced without IL-2, augmented the growth of early erythroid progenitors, the LAK cell fraction did not. This fraction suppressed colony growth from mature granulocyte-macrophage progenitors (day 7 CFU-GM) especially with an 18-h preincubation prior to coculture. It also suppressed both immature granulocyte-macrophage progenitors (day 14 CFU-GM) and multipotential hematopoietic progenitors. The suppressive effects were observed on colony growth from autologous marrow cells as well as allogeneic marrow cells. The suppression of day 7 CFU-GM colony growth by supernatants due to preincubation with marrow cells and the LAK cell fraction suggested that the humoral factor contributes to the suppression by the LAK cell fraction. These data suggest that the LAK cell fraction suppresses the development of human hematopoietic progenitor cells.     

Transformation of early erythroid precursor cells (BFU-E) by a recombinant murine retrovirus containing v-erb-B

Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that transforms erythroid and fibroblast cells in vitro and in vivo. The transforming ability of AEV is due primarily to the oncogene v-erb-B. A recombinant murine retrovirus has been constructed by inserting a chimeric gag-v-erb-B gene into a Moloney murine leukemia virus based vector. This retrovirus was used to examine v-erb-B-induced transformation of murine hematopoietic cells. Infection of murine primary fetal liver, adult bone marrow or adult spleen cells with the recombinant virus generated large hemoglobinized erythroid colonies in the absence of exogenous growth factors. Generation of such colonies usually requires the presence of erythropoietin (Epo) and interleukin-3 (IL-3). These growth-factor independent colonies were shown to be derived from early (BFU-E) and not late (CFU-E) erythroid progenitor cells, and the effect was not attributable to growth factors elicited by the virus-producing cell lines. In order to confirm that the recombinant virus was responsible for this transformation of BFU-E to growth factor independence, bone marrow cells from post 5-fluorouracil treated mice were infected and used to repopulate lethally-irradiated mice. Growth factor-independent BFU-E were obtained in up to 30% of day-13 spleen colonies and it was shown by DNA analysis that cells from these colonies contained integrated provirus. Our results indicate that v-erb-B transforms early erythroid progenitors to growth factor independent growth and subsequent differentiation to erythrocytes -a process that normally requires Epo plus either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF).     

c-abl function in normal and chronic myelogenous leukemia hematopoiesis: in vitro studies with antisense oligomers.

By using antisense oligomers the functional role of the c-abl proto-oncogene in the in vitro growth of bone marrow hematopoietic progenitors from normal subjects and patients with chronic myelogenous leukemia (CML) has been evaluated. Light density bone marrow cells (LDBMs) were depleted of adherent cells, pre-incubated for 15 h with the appropriate oligomer at a concentration of 14 microns, and then plated in methylcellulose for the evaluation of colony formation. Both anti-exon Ia and anti-exon Ib antisense oligomers produced a significant inhibition of normal day 14 CFU-GM growth in vitro (n = 5, 41 +/- 11%, and 36 +/- 7%, respectively; p less than 0.01). In contrast, normal BFU-E growth was not significantly influenced by antisense oligomers (n = 5, 14 +/- 21% and 7 +/- 19%, respectively; p less than 0.05). These findings were confirmed by plating CD34 positive progenitors. When interleukin 3 (IL-3) (100 ng/ml) was added to the culture medium during the preincubation of LDBMCs, the inhibitory effects of antisense oligomers on normal CFU-GM growth were abolished. Seven patients with CML were also studied, all of whom had cytogenetic evidence of 100% clonal hematopoiesis. In five patients in the chronic phase, antisense oligomers were inhibitory on in vitro growth of both day 14 CFU-GM (37 +/- 20% and 37 +/- 15%, p less than 0.05) and BFU-E (45 +/- 15% and 41 +/- 11%, p less than 0.05), and this inhibition was not removed by pre-incubation with IL-3. No significant effect was observed on cluster or colony formation in two patients with CML in accelerated or blastic phase, and on in vitro growth of clonogenic cells from the Ph1-positive K-562 cell line. These findings (i) confirm previous observations showing a lineage specific requirement of c-abl function in normal hematopoiesis, and (ii) suggest that the residual c-abl expression has a role in chronic phase CML hematopoiesis, as its inhibition impairs both myeloid and erythroid colony formation in vitro.     

The role of marrow accessory cell populations in the augmentation of human erythroid progenitor cell (BFU-E) proliferation by prostaglandin E

The requirement for CD8+ T lymphocytes in the stimulation of erythroid progenitor cells by prostaglandin E (PGE) was examined. When low density bone marrow (LD-BM) or non-adherent bone marrow (NA-BM) cells were depleted of CD8+ cells the enhancing effect of PGE on BFU-E proliferation was abrogated. However, further enrichment of marrow progenitor cells by depletion of accessory cells using a cocktail of specific monoclonal antibodies, immunoadherence and fluorescence activated cell sorting with the MY10 monoclonal antibody resulted in a population of erythroid progenitor cells which were responsive to the enhancing effect of PGE despite the absence of CD8+ cells. Stepwise individual cell lineage depletion of marrow cell populations indicated that prostaglandin E enhanced erythroid burst formation in the absence of CD8+ cells provided that glycophorin-A+ cells were removed from LD-BM or NA-BM cells. These results suggest that nucleated erythroid cell populations may modulate the enhancement of BFU-E by PGE. The ability of GP-A+ cells to block the enhancement of erythroid burst formation by PGE following removal of CD8+ T cells was confirmed by readdition of conditioned medium prepared from positively selected GP-A+ marrow cells. These results expand the role of CD8+ T cells in the PGE enhancement of BFU-E proliferation and suggest another mechanism by which accessory cells regulate the proliferation of BFU-E in bone marrow.     

Suppression of T-cell response in autologous mixed lymphocyte-tumor culture by large granular lymphocytes

The role of large granular lymphocytes (LGL) in the autologous mixed lymphocyte-tumor culture (MLTC) was studied in cancer patients with malignant pleural effusions. When blood lymphocytes were cocultured in vitro with autologous tumor cells freshly isolated from carcinomatous pleural effusions, [3H]thymidine incorporation was weakly stimulated on day 6 in 6 of 30 samples. Removal of LGL from the responder population by treatment with the Leu-7 or Leu-11b monoclonal antibody plus complement (C') induced or augmented the proliferative response. LGL and small T-lymphocytes were isolated by discontinuous Percoll gradient centrifugation and tested separately for the proliferative response to autologous tumor. T-cells proliferated in 24 of 30 cases, while LGL showed no proliferation. Addition of LGL to autologous mixed T-cell-tumor cultures suppressed the proliferation of T-cells. LGL, however, did not inhibit T-cell proliferation induced by alloantigens and lectins. The suppressive activity of Percoll-purified LGL was not reduced by OKT3 plus C' treatment, but it was totally abrogated by Leu-11b plus C'. Supernatants produced by 24-hour culture of LGL with autologous tumor contained soluble factors that suppressed the autologous MLTC without killing the autologous tumor or T-blasts. The LGL-mediated suppression was not abolished by anti-interferon-alpha or anti-interferon-gamma antibody. Activation of T-cells in the autologous MLTC induced lytic potential restricted to autologous tumor. In the presence of LGL, T-cells failed to develop autotumor killing activity. Once autotumor killer T-cells were generated in autologous MLTC, their cytotoxicity was no longer inhibited by LGL. These results indicate that LGL from patients with carcinomatous pleural effusions suppress the capacity of autotumor-recognizing T-lymphocytes to proliferate and develop autotumor cytotoxicity in the autologous MLTC. This could explain why fresh T-cells have no cytolytic activity to autologous tumor.     

The prognostic value of in vitro cultures of erythroid and megakaryocyte progenitors in myelodysplastic syndromes

The prognostic value of colony formation by granulocyte-macrophage progenitors (CFU-GM) in myelodysplastic syndromes (MDS) has been investigated in several studies. We studied the in vitro growth patterns of hematopoietic progenitors of 83 patients with an MDS to find out whether erythroid (BFU-E) and megakaryocyte (CFU-Meg) cultures yield additional prognostic information to that obtained with CFU-GM cultures. Thirty-nine of 82 patients showed normal CFU-GM colony formation; the others had either excessive growth of colonies/clusters or reduced growth. Five of 74 patients had normal BFU-E and nine of 39 patients normal CFU-Meg growth; the others showed reduced or absent colony formation. The cultures of each cell lineage had a similar prognostic impact: the patients with a normal growth pattern had a lower risk of developing leukemia and a longer survival than those with an abnormal growth pattern (significant difference or trend). All patients with normal BFU-E or CFU-Meg colony growth also had normal CFU-GM colony formation, and all patients with normal BFU-E growth also had normal CFU-Meg growth. Among the patients with normal CFU-GM cultures, those with normal erythroid or megakaryocyte colony formation had a trend towards a better outcome compared to those with an abnormal growth pattern in the same cell lineage. In conclusion, erythroid and megakaryocyte cultures did not significantly contribute to the prognostic information obtained with CFU-GM cultures in MDS.     

Expression of HLA class II determinants by normal and chronic myeloid leukemia progenitors

It has been suggested that the expression of some HLA class II antigens, derived from three loci (DR, DP, DQ) is important in the regulation of both the immune response and the response of haemopoietic progenitors to regulation factors, such as acidic isoferritins (AIF), as well as in the interaction between T lymphocytes and erythroid progenitors (BFU-E). Changes in the expression of class II antigens have been reported on the surface of granulo-monocyte progenitors in chronic myeloid leukemia (CML) and correlated to the abnormal proliferation of such cells. In this study, monoclonal antibodies against DR and DQ monomorphic determinants were used to investigate the expression of these antigens on the surface of normal and CML bone marrow and peripheral blood BFU-E by means of complement mediated cytotoxicity. It was found that most normal and leukemic BFU-E express DR antigens. Antigens density tends to be greater on marrow as opposed to peripheral precursors. In addition, leukemic BFU-E are more sensitive to cytolytic treatment than their normal counterparts. Normal BFU-E do not express detectable amounts of DQ antigens, whereas these are present on a proportion of leukemic BFU-E.     

Anemia associated with feline leukemia virus infection in cats.

The types of anemia associated with natural and experimental feline leukemia virus (FeLV) infection in cats were investigated. In one experiment, 10 kittens were inoculated neonatally with Jarrett FeLV-1, an isolate of subgroup A; 6 developed anemia a few weeks later. This anemia was characterized by macrocytosis, normoblastosis, increased erythropoiesis in the bone marrow, and extramedullary hematopoiesis in the spleen. Anemia was transient and nonfatal and occurred before the onset of lympoid malignancy. The same type of anemia was also seen in 9 of 24 kittens inoculated with Jarrett FeLV-9 of subgroups A and B. A different form of anemai occurred in another experiment in which 10 kittens were inoculated with FeLV-C of subgroup C only. All 10 kittens developed a profound aplastic or erythroblastopenic anemia in which the bone marrow became depleted of erythroid tissue; all kittens died within 16 weeks, most as a direct result of anemia. In an experiment in which kittens were inoculated with FeLV-B of subgroup B only, no kitten showed anemia. Cats with naturally acquired, nonleukemic lymphosarcoma were also studied. Of 33 lymphosarcomas in which myelophthisis was excluded as a cause, 54% of the affected cats had anemia, the features of which were consistent with hemolytic origin. When virus could be grown from these lymphosarcomas, it was of subgroup A alone or a combination of A and B. With one exception, anemic cats had low or negative titers to feline oncornavirus-associated cell membrane antigens. Until more isolates have been tested, it is not known if the various hematologic changes reflected differences in the pathogenic effects of the subgroups of the virus or of types of strains within them.     

Angiotensin-(1–7) synergizes with colony-stimulating factors in hematopoietic recovery

Purpose

Angiotensin (1–7) [A(1–7)] is a bioactive peptide of the renin angiotensin system that stimulates the number of bone marrow progenitors and hematopoietic recovery after myelosuppression. We evaluated the combination of A(1–7) with colony-stimulating factors, Neupogen and Epogen, on bone marrow progenitors and the recovery of circulating formed elements following chemotherapy.

Methods

Mice were injected with gemcitabine followed 2 days later with A(1–7). Circulating blood cells and bone marrow progenitors were measured over time.

Results

Combination of A(1–7) with Neupogen (the latter given only 3 days starting at the white blood cell nadir) decreased the amount of Neupogen needed for optimal recovery by 10-fold. The progenitors measured include CFU-GEMM, CFU-GM, CFU-Meg and BFU-E. A(1–7) increased recovery of all progenitors when given alone or in combination with Neupogen above that with Neupogen alone. Combination of A(1–7) with Epogen slightly increased (not significantly) red blood cell concentrations above those achieved by Epogen alone. However, in this model, A(1–7) or A(1–7) in combination with Epogen increased all erythroid progenitors with the largest effect on early erythroid progenitors (immature BFU-E).

Conclusions

Neupogen and Epogen acted synergistically with A(1–7) to increase the concentration of myeloid, megakaryocytic and erythroid progenitor cells in the bone marrow following chemotherapy suggesting that A(1–7)’s multilineage effect on early progenitors in the marrow facilitates proliferation in response to lineage-specific growth factors.     

The comparative enhancing effects of prostaglandin E1 on colony formation by erythroid progenitor (BFU-E) cells from bone marrow of mice differing in the Fv-2 locus

Prostaglandin E1(PGE1) was assessed for its colony enhancing effects on erythroid (BFU-E) progenitor cells and for its colony suppressive effects on granulocyte-macrophage (CFU-GM) progenitor cells from bone marrows of mice differing in the Fv-2 locus. The Fv-2 locus has been reported by others to control the proportion of BFU-E, but not of CFU-GM, in DNA synthesis. PGE1 significantly enhanced erythroid colony formation by marrow cells from DBA/2 (Fv-2ss), C57BL/6 (Fv-2rr) and BDF1 (Fv-2rs) mice, but the DBA/2 cells were more sensitive to the PGE1 enhancing effects than were cells from C57BL/6 or BDF1 mice. The enhanced sensitivity of DBA/2 cells to PGE1 was associated with the higher cycling rate of DBA/2-BFU-E in comparison with C57BL/6- and BDF1-BFU-E, and removal of S-phase DBA/2-BFU-E by pulse exposure of cells to high specific activity tritiated thymidine in vitro eliminated the erythroid colony enhancing effects of PGE1. The differences in sensitivity of BFU-E from mice differing in the Fv-2 locus to the effects of PGE1 were verified using Fv-2 congenic mice. In contrast, no significant differences were noted in sensitivity of CFU-GM from these different mouse strains, including mice congenic for the Fv-2 locus, to the suppressive effects of PGE1. This correlated with the similar cycling characteristics of CFU-GM from these mice, and the non-cycle specific effects of PGE1 on mouse CFU-GM. These studies substantiate further the regulatory effects of the Fv-2 locus on mouse erythroid progenitor cells which are a manifestation of the cycling rates of these cells.     

Effects of recombinant human tumor necrosis factor on highly enriched hematopoietic progenitor cell populations from normal human bone marrow and peripheral blood and bone marrow from patients with chronic myeloid leukemia

Previous studies using unseparated normal human bone marrow cells have indicated that recombinant tumor necrosis factor alpha (rTNF-alpha) can inhibit the in vitro colony growth by normal granulocyte/macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells in a dose-dependent manner. In the present studies, by using very low numbers of highly enriched normal bone marrow progenitor cell populations as target cells, we have extended these previous findings to provide convincing evidence that erythroid and myeloid colony growth suppression by rTNF-alpha is manifested by a direct interaction between rTNF-alpha and CFU-GM and BFU-E progenitor cells. In addition, the sensitivity of normal peripheral blood and chronic myeloid leukemia bone marrow CFU-GM and BFU-E colony growth to inhibition by rTNF-alpha was examined and found to be comparable with that of normal bone marrow CFU-GM and BFU-E. Although the continuous presence of high doses of rTNF-alpha (5000 units/ml) was required in methylcellulose cultures for maximal CFU-GM (90%) and BFU-E (70%) colony suppression, short-term exposure (24 to 72 hr) of normal bone marrow-enriched progenitor cells to rTNF-alpha, in the absence of hematopoietic growth factors, was sufficient to irreversibly suppress up to 50 to 65% of CFU-GM colony growth. In contrast, the number of BFU-E colonies was increased under these conditions. If, however, hematopoietic growth factors (Mo-T-cell-conditioned medium and erythropoietin) were present during preincubation of the cells with rTNF-alpha, BFU-E were then slightly suppressed while the extent of CFU-GM inhibition remained essentially the same. The suppressive effect of rTNF-alpha on erythroid and myeloid progenitor cell growth appears to be most pronounced on the more primative stages of committed progenitor cell development, since inhibition of CFU-GM- and BFU-E-derived colony growth progressively decreased with the delayed addition of rTNF-alpha to methylcellulose cultures. [3H]Thymidine incorporation was also inhibited by rTNF-alpha in normal bone marrow-enriched progenitor cell populations stimulated to proliferate in liquid culture by colony-stimulating factors. This effect was transient, however, since the activity of rTNF-alpha declined after the first 24 h of culture at 37 degrees C, particularly at low doses of rTNF-alpha where the activity was completely lost after 48 h of culture. This loss of activity appeared to be due to a decreased sensitivity of progenitor cells to the antiproliferative effects of tumor necrosis factor (TNF) after an initial exposure rather than a lack of available TNF.(ABSTRACT TRUNCATED AT 400 WORDS)