Chronic childhood neutropenia: studies on the in vitro clonogenicity of bone marrow myeloid progenitors

Bone marrow mononuclear cells (MNC) from 6 pediatric patients with chronic neutropenia were tested for myeloid colony formation upon stimulation with the supernatant of the 5637 cell line or with recombinant granulocyte-macrophage colony-stimulating factor or interleukin 3. Heterogeneous patterns of response of myeloid progenitors were observed in the individual patients, with no colony growth in 2 cases and abnormalities of colony formation or composition in two additional cases. Morphologic and surface marker analyses showed that the bone marrow of some patients contained an excess of lymphocytes with an altered subset distribution. In order to investigate whether or not there was a relationship between the latter abnormality and the observed clonogenic defects, marrow MNC were tested for myeloid colony formation before and after lymphocyte depletion. No evidence for a cell-mediated suppression of colony growth was obtained; likewise, patient sera failed to inhibit colony formation by normal bone marrow myeloid progenitors. Taken together, these data make it unlikely that, in our cases, immunologic mechanisms were responsible for the pathogenesis of chronic neutropenia.     

Augmentation of donor bone marrow engraftment in histoincompatible murine recipients by granulocyte/macrophage colony-stimulating factor

T cell depletion of donor bone marrow can prevent graft v host disease (GVHD) in human and murine marrow graft recipients. However, engraftment in the recipient may be compromised as a consequence of donor marrow T cell depletion. The effect of recombinant murine granulocyte/macrophage colony-stimulating factor (rmu GM-CSF) on engraftment and hematologic reconstitution was evaluated in a murine allogeneic bone marrow transplantation (BMT) model involving T cell depletion of marrow. Before transplantation into irradiated mice differing at major and minor histocompatibility loci, rmu GM-CSF was preincubated with T cell-depleted donor marrow. When low degrees of engraftment were noted in control recipients, treatment of donor marrow with high concentrations of rmu GM-CSF led to enhanced engraftment. Ex vivo donor graft incubation with rmu GM-CSF or a single in vivo injection of rmu GM-CSF were both effective means of promoting engraftment. When the engraftment rate in control recipients was high, rmu GM-CSF did not have an identifiable effect. Only slight increases in hematologic recovery were detected regardless of the rate of engraftment. Neither post-BMT survival nor marrow stem cell capacity was affected by rmu GM-CSF incubation. Furthermore, growth factor administration did not have a significant effect on the incidence of GVHD in recipients of non-T cell-depleted bone marrow splenocyte preparations. In vitro natural killer-mediated target cell lysis was not altered by incubation of effector cells with rmu GM-CSF. These results demonstrate the potential of ex vivo rmu GM-CSF treatment of donor marrow to facilitate engraftment across extensive histo- compatibility barriers.     

In vitro studies on the radiosensitivity of multipotent hemopoietic progenitors in canine bone marrow

The in vitro radiation response to 280-kV x-rays (does rate 72 cGy/min) of multipotent hemopoietic progenitor cells, mixed colony-forming units (CFU-mix), from canine bone marrow was assayed and compared to the radiation response characteristics of early erythroid progenitors, erythroid burst-forming units (BFU-E). To improve the colony-forming efficiency, the effect of various bone marrow cell separation techniques on colony formation of both progenitors was examined. The separation of bone marrow aspirates by discontinuous buoyant gradient centrifugation using the lymphocyte separation medium Lymphoprep with a density of 1.070 g/ml allowed the establishment of reproducible survival curves. The survival curves for both progenitors were strictly exponential, and CFU-mix were found to be more radiosensitive (D0 = 12 +/- 2 cGy) than BFU-E (D0 = 16 +/- 2 cGy).     

Quantitative development of adherent cell colonies in bone marrow cell culture in vitro.

Quantification of the formation of adherent cell colonies in bone marrow cell culture was attempted. By secondary transfer of the bone marrow cells as a single cell suspension after 4 days' culture of fine marrow fragments, a linear relationship was obtained between the number of adherent cell colonies developing and the number of cells secondarily inoculated into the culture bottle. This suggests that 4 days' culture of the bone marrow cells with close intercellular interactions is sufficient for the 'conditioning' of the cells to develop adherent cell colonies. Activity of such colonies to support haemopoietic stem cell proliferation was also shown.     

Allogeneic bone marrow transplantation: the monitoring of granulocyte macrophage colonies following the collection of bone marrow mononuclear cells and after the subsequent in-vitro cytolysis of OKT3 positive lymphocytes

Marrow nucleated cells from eight normal allogeneic donors was layered on Ficoll-Metrizoate to isolate the mononuclear cell fraction. The cells were then washed to remove Ficoll-Metrizoate and coagulation factors prior to resuspension in a balanced salt solution and the addition of the murine anti-human T-lymphocyte monoclonal antibody OKT3 and rabbit complement. The procedures were assessed for their effect on mononuclear cell viability (mean recovery 84.4%); the ability of the cells to proliferate granulocyte-macrophage colonies (mean recovery 57.4%); the in-vitro T-lymphocytolysis (mean 75.7%) and the removal of rabbit complement (greater than 99%). Following marrow transplantation with this treated mononuclear fraction the mean day to recovery of greater than or equal to 1.0 X 10(9)/l leucocytes was 20 d, with three patients developing greater than or equal to Grade II acute graft versus host disease (GvHD). Thus, treatment of donor marrow with OKT3 and complement in a large volume system was not detrimental to subsequent engraftment, nor effective in complete T-lymphocytolysis, nor in prevention of severe GvHD.     

Polycythemia and the Budd-Chiari syndrome: study of serum erythropoietin and bone marrow erythroid progenitors

The mechanism of polycythemia associated with the Budd-Chiari syndrome is unknown. Erythropoiesis in 10 patients with Budd-Chiari syndrome was studied in an attempt to distinguish prior unrecognized polycythemia vera from secondary polycythemia. Serum erythropoietin was assayed using a mouse fetal liver erythroblast assay. High concentrations of serum erythropoietin were observed in 6 of 7 patients with acute primary Budd-Chiari syndrome. Levels were normal in four patients who were investigated during the chronic phase and were increased in one with persisting polycythemia. In one patient, erythropoietin concentration in the hepatic vein was twice the level measured in peripheral, caval and renal venous blood. Bone marrow erythroid progenitors developed in vitro in the absence of exogenous erythropoietin in all polycythemia vera cases studied in acute and chronic phases, whether polycythemia persisted or not. These findings indicate that hepatic erythropoietin production occurs in the acute phase of Budd-Chiari syndrome and suggest that, in some cases of Budd Chiari syndrome, polycythemia which resolves after the acute phase may be secondary to liver disease.     

A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: significant induction of peripheral and bone marrow neutrophilia

Host defenses in the human neonate are limited by immaturity in phagocytic immunity. Such limitations seem to predispose infected newborns to neutropenia from an exhaustion of the neutrophil reserve. Among the critical defects thus far identified in neonatal phagocytic immunity is a specific reduction in the capacity of mononuclear cells to express granulocyte colony-stimulating factor (G-CSF) after stimulation. However, the safety, pharmacokinetics, and biological efficacy of administration of recombinant human (rh)G-CSF to infected human newborns to compensate for this deficiency is unknown. Forty-two newborn infants (26 to 40 weeks of age) with presumed bacterial sepsis within the first 3 days of life were randomized to receive either placebo or varying doses of rhG-CSF (1.0, 5.0 or 10.0 micrograms/kg every 24 hours [36 patients] or 5.0 or 10.0 micrograms/kg every 12 hours [6 patients]) on days 1, 2, and 3. Complete blood counts with differential and platelet counts were obtained at hours 0, 2, 6, 24, 48, 72, and 96. Circulating G-CSF concentrations were determined at hours 0, 2, 6, 12, 14, 16, 18, 24, and 36. Tibial bone marrow aspirates were obtained after 72 hours for quantification of the bone marrow neutrophil storage pool (NSP), neutrophil proliferative pool, granulocyte progenitors, and pluripotent progenitors. Functional activation of neutrophils (C3bi expression) was determined 24 hours after rhG-CSF or placebo administration. Intravenous rhG-CSF was not associated with any recognized acute toxicity. RhG-CSF induced a significant increase in the blood neutrophil concentration 24 hours after the 5 and 10 micrograms/kg doses every 12 and 24 hours and it was sustained as long as 96 hours. A dose-dependent increase in the NSP was seen following rhG-CSF. Neutrophil C3bi expression was significantly increased at 24 hours after 10 micrograms/kg every 24-hour dose of rhG- CSF. The half-life of rhG-CSF was 4.4 +/- 0.4 hours. The rhG-CSF was well tolerated at all gestational ages treated. The rhG-CSF induced a significant increase in the peripheral blood and bone marrow absolute neutrophil concentration and in C3bi expression. Future clinical trials aimed at improving the outcome of overwhelming bacterial sepsis and neutropenia in newborn infants might include the use of rhG-CSF.     

Synergistic inhibition in vivo of bone marrow myeloid progenitors by myelosuppressive chemokines and chemokine-accelerated recovery of progenitors after treatment of mice with Ara-C

OBJECTIVE: Selected chemokines suppress proliferation of hematopoietic progenitor cells (HPCs) in vitro; some of these have demonstrated inhibition of myelopoiesis in vivo. Because myelosuppressive chemokines synergize in vitro with other myelosuppressive chemokines, we sought to determine whether additional chemokines active in vitro were myelosuppressive in vivo and whether combinations of myelosuppressive chemokines synergized in vivo to dampen myelopoiesis. We also evaluated three chemokines in vivo for myeloprotection against Ara-C-induced decreases in HPCs. METHODS: C3H/HeJ mice were used for analysis of in vivo influence of chemokines, with the end points being effects on absolute numbers and cycling status of HPCs. RESULTS: When used alone, CCL2, CCL3, CCL19, CCL20, CXCL4, CXCL5, CXCL8, CXCL9, and XCL1 caused dose-dependent significant decreases in absolute numbers and cycling status of HPCs in vivo. The following combinations of two chemokines resulted in in vivo myelosuppression at concentrations much lower than that induced by each chemokine alone: CCL3 plus either CXCL8 or CXCL4, CXCL8 plus CXCL4, CCL2 plus either CCL20 or CXCL9, CCL20 plus CXCL9, CXCL5 plus either XCL1 or CCL19, XCL1 plus CCL19, and CCL3 plus CCL19. Also, mice injected with CXCL8, CXCL4, or the chimeric CXCL8/CXCL4 protein CXCL8M1 manifested accelerated recovery of absolute numbers of HPCs in response to the toxic effects of Ara-C administration. CONCLUSIONS: A number of chemokines shown previously to manifest inhibitory effects in vitro for proliferation of HPCs are now demonstrated to also induce myelosuppression in vivo. Moreover, combinations of low dosages of two myelosuppressive chemokines when administered together demonstrate synergistic suppression in vivo. Additionally, chemokines, including a CXCL8M1 chimeric protein previously shown to manifest enhanced suppression of HPC proliferation in vitro and in vivo, accelerate HPC recovery after treatment of mice with Ara-C. These results may be of use for future clinical utility of chemokines in a myelosuppressive/myeloprotective setting.     

Granulocytic and stromal progenitors in the bone marrow of patients with primary myelofibrosis

Quantitative and qualitative changes in granulocyte-macrophage (CFU-GM) and fibroblast colony-forming cells (CFU-F) were studied in 7 patients with primary myelofibrosis (MF). Marrow cells were collected from bone biopsy specimens after treatment with collagenase. The number of CFU-GM correlated with the amount of haemopoietic tissue noted in the bone marrow histology and ranged between 0-400/mg of bone. CFU-F were increased in 2 patients with moderate fibrosis. Circulating CFU-GM were increased in all patients studied (169-3749/ml of blood). There was no significant correlation between the number of CFU-GM in the bone marrow and that in the blood. Cytochemical studies showed a high incidence in eosinophil progenitors in the bone marrow and especially in the blood of patients with MF. These data suggest a functional abnormality of myeloid progenitors in this disease.     

白细胞介素—11对CITP患者骨髓巨核细胞的影响

目的：观察重组人白细胞介素11（rhIL-11)对慢性特发性血小板减少性紫癜（CITP）患者骨髓巨核祖细胞增殖和成熟的影响。方法：在21例（CITP患者骨髓巨核祖细胞培养体系中加入不同浓度的rhIL-11,用血浆凝块法培养14d后,用ABC试剂盒染色观察集落生长数,用MCDS－2010骨髓细胞分析系统测定巨核细胞的面积、直径;15例健康人作对照组。结果：CITP组CFU－MK、BFU－MK总集落数较对照组略低（P＞0．05）,而ITP组巨核细胞面积、直径均低于对照组（P＜0．05）;不同浓度rhIL-11在体外对CITP组及对照组的巨核组细胞集落生成及成熟度均有促进作用,呈剂量依赖性,以100μg/L作用最佳;骨髓涂片巨核细胞数与CFU－MK及总集落数呈正相关（P＜0．05）,BFU－MK、面积、直径与巨核细胞数无相关性（P＞0．05）。结论：CITP患者的骨髓巨核细胞存在成熟障碍。rhIL-11对CITP患者的骨髓巨核祖细胞的增殖与成熟度均有促进作用,并呈剂量依赖性。     

Megakaryocyte progenitors in the bone marrow and peripheral blood of patients with myeloproliferative diseases

We studied the behavior in culture of megakaryocyte progenitor cells (CFU-Mk) from peripheral blood (PB) and bone marrow (BM) cells in eight patients with myeloproliferative diseases (MPD). In seven patients we observed megakaryocyte (Mk) colony formation from PB cells, which were generated in the absence of any added stimulator and which did not increase after the addition of a source of Mk-colony stimulating activity (CSA-Mk). The number of BM CFU-Mk was significantly higher in patients than in controls, and in seven out of eight patients the responsiveness to added CSA-Mk was retained. Plasma obtained from six patients did not stimulate normal donors' BM target cells to form Mk colonies. These data demonstrate an expansion of the CFU-Mk pool in MPD patients without increased plasma levels of CSA-Mk, and suggest that PB and BM CFU-Mk of MPD patients might have different kinetic properties.     

Aplastic anemia: evidence for dysfunctional bone marrow progenitor cells and the corrective effect of granulocyte colony-stimulating factor in vitro

We investigated the effects of granulocyte-macrophage colony- stimulating factor, interleukin-3, stem cell factor, interleukin-6, and granulocyte colony-stimulating factor (G-CSF) alone, and in combination, on the clonogenic potential of normal and aplastic anemia (AA) bone marrow mononuclear cells (BMMC and CD34+ cells. AA BMMC consistently produced a significantly lower absolute number of colonies than normal, but, when account was taken of the reduced proportion of CD34+ cells in AA BM, there was no significant difference in terms of cloning efficiency (CE). However, when removed from the influence of accessory cells, the CE of AA CD34+ cells decreased significantly more than normal, indicating a defect in their function, either in terms of dependence on accessory cell-derived factors or susceptibility to cell damage when sorted. Of the factors studied, G-CSF had the most significant effect on the response of CD34+ cells from both groups when removed from their accessory cells. This was particularly true for AA CD34+ cells, whose response to cytokine stimuli containing G-CSF enabled them to match the response of normal CD34+ cells.     

Expression of DR antigens on early human progenitors of neutrophilic and eosinophilic colonies in diffusion chambers (CFUd)

Incubation of the non-adherent low density cells from normal human bone marrow with an antiserum to DR antigens markedly inhibited their capacity to form granulocytic colonies in fibrin clot diffusion chambers implanted intraperitoneally into irradiated mice. The inhibitory effect of antiserum was abrogated after absorption of antiserum with B cells. Inhibition was obtained both with and without complement treatment. Neutrophilic colony formation exhibited a higher sensitivity to diluted antiserum to DR antigens as compared with eosinophilic colony formation. These results suggest that DR determinants are expressed on the neutrophilic and eosinophilic progenitor cells (CFUd) which give rise to colonies in diffusion chambers.