Marrow Mononuclear Cells Research Articles

Nucleotide and prostaglandin cycling in canine cyclic hematopoiesis.

Bone marrow cells were collected from normal dogs, normal dogs made neutropenic with cyclophosphamide, and 11 dogs affected with cyclic hematopoiesis (CH) on 3 consecutive days of separate 12- to 14-day cycles. The mononuclear marrow cells from both groups of control dogs and from the CH dogs on each of 12-cycle days were cultured for 2.5 hr in serum-free media. The amounts of prostaglandins (PGF2 alpha and PGE) and cyclic GMP (cGMP) measured in the media were found to vary with the cycle in the CH dog. PGF2 alpha was highest as the dogs recovered from the neutropenia and lowest 4 days before the onset of the next neutropenic episode. Cyclic GMP was lowest 4-5 days before the onset of neutropenia, then dramatically increased as the neutropenic period approached. Cyclic GMP was highest when PGF2 alpha was lowest. Normal dogs, made neutropenic with a single dose of cyclophosphamide, had elevations of PGF2 alpha but not PGE or cGMP during the recovery period of active granulopoiesis.

Unusual T cell proliferations and neutropenia in rheumatoid arthritis: comparison with classical Felty's syndrome

4 patients are described with rheumatoid arthritis, splenomegaly, neutropenia and an unusual proliferation of T cells in the blood and marrow. These patients are clinically similar to patients with classical Felty's syndrome but can be distinguished from them by staining blood and marrow mononuclear cells with a panel of monoclonal anti-T cell antibodies. The T cells from patients with T cell proliferations stain with UCHT1 (OKT3 equivalent) and UCHT4 (OKT8 equivalent but do not stain with a panel of OKT1-like antibodies. In 7 patients with classical Felty's syndrome there was no increase of UCHT4 cells in the blood and the large majority of T cells stained with OKT1-like antibodies. The marrows from the patients with T cell proliferations contain plentiful haemopoietic progenitor cells and it is probable that the T lymphocytes suppress their normal maturation. There was a poor response to splenectomy in 2 patients with T cell proliferations, and single cytotoxic drug therapy may be more appropriate when therapy is required. The literature is reviewed and it is suggested that the T cell proliferations may be secondary to the rheumatoid process.

Selective Increases of Procoagulant Activity in Rabbit Lymphoid Populations In Vitro Following Stimulation with Endotoxin: Dependence on Anatomic Source

Rabbit mononuclear leukocytes isolated from a variety of anatomic sites were examined for ability to generate procoagulant activity in vitro. Marrow, blood and spleen mononuclear cell populations were found to differ functionally from lymph node, thymus and alveolar populations by having much greater ability to increase in tissue factor activity in response to an endotoxin stimulus. Thus, after incubation in the presence of endotoxin, leukocytes obtained from marrow, blood, and spleen were found to increase in procoagulant activity characterized as tissue factor by 832, 1942 and 12.6 fold, respectively. In contrast, pulmonary alveolar macrophages increased in tissue factor activity only by 2.8 fold, and lymph node and thymus mononuclear cells showed little or no increases. These functional differences, demonstrated by exposing the six cell populations to endotoxin under controlled conditions in vitro, likely explain the similar pattern of anatomic selectivity of leukocyte tissue factor increases reported to occur in vivo during endotoxemia and Shwartzman reactions (1).

Cholesterol and Mevalonic Acid Are Independent Requirements for the In Vitro Proliferation of Human Bone Marrow Granulocyte Progenitor Cells: Studies Using ML-236B

ML-236B is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the key regulatory enzyme in the sequence that catalyzes the conversion of acetate to mevalonic acid in cholesterol biosynthesis. This compound caused marked inhibition of human bone marrow granulocyte progenitor cell (CFU-C) proliferation, the 50% inhibitory concentration (IHD50) being 2.0 X 10(6)M. Inhibition of colony formation was reversed by mevalonic acid but not by cholesterol. ML-236B also inhibited DNA synthesis and acetate incorporation into cholesterol in marrow mononuclear cells (IHD50 = 5.6 x 10(6)M and 3.2 x 10(7)M, respectively). No inhibition of mevalonate incorporation into cholesterol was observed. These results differ from those observed with 25-hydroxycholesterol, another inhibitor of HMG CoA reductase. The latter compound also inhibited CFU-C proliferation and cholesterol biosynthesis from acetate; inhibition of colony formation was reversed by cholesterol but not by mevalonic acid. In addition, 25-hydroxycholesterol inhibited cholesterol synthesis from mevalonic acid precursor. We conclude that: (1) ML-236B is a potent inhibitor of CFU-C proliferation, DNA synthesis, and cholesterol biosynthesis from acetate precursor in marrow mononuclear cells; (2) the effects of ML-236B are completely reversed by mevalonic acid but not by cholesterol, suggesting that mevalonic acid per se or one or more of its nonsterol products are critical for cell growth; (3) the inhibitory effects of 25-hydroxycholesterol on CFU-C proliferation and cholesterol biosynthesis are not solely a result of its inhibition of HMG CoA reductase, but are due in part to inhibition of enzymatic steps distal to mevalonic acid in the sterol synthetic pathway; and (4) mevalonic acid and cholesterol are independent requirements for CFU-C proliferation and differentiation in vitro.

Cholesterol and mevalonic acid are independent requirements for the in vitro proliferation of human bone marrow granulocyte progenitor cells: studies using ML-236B

ML-236B is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the key regulatory enzyme in the sequence that catalyzes the conversion of acetate to mevalonic acid in cholesterol biosynthesis. This compound caused marked inhibition of human bone marrow granulocyte progenitor cell (CFU-C) proliferation, the 50% inhibitory concentration (IHD50) being 2.0 X 10(6)M. Inhibition of colony formation was reversed by mevalonic acid but not by cholesterol. ML-236B also inhibited DNA synthesis and acetate incorporation into cholesterol in marrow mononuclear cells (IHD50 = 5.6 x 10(6)M and 3.2 x 10(7)M, respectively). No inhibition of mevalonate incorporation into cholesterol was observed. These results differ from those observed with 25-hydroxycholesterol, another inhibitor of HMG CoA reductase. The latter compound also inhibited CFU-C proliferation and cholesterol biosynthesis from acetate; inhibition of colony formation was reversed by cholesterol but not by mevalonic acid. In addition, 25- hydroxycholesterol inhibited cholesterol synthesis from mevalonic acid precursor. We conclude that: (1) ML-236B is a potent inhibitor of CFU-C proliferation, DNA synthesis, and cholesterol biosynthesis from acetate precursor in marrow mononuclear cells; (2) the effects of ML-236B are completely reversed by mevalonic acid but not by cholesterol, suggesting that mevalonic acid per se or one or more of its nonsterol products are critical for cell growth; (3) the inhibitory effects of 25- hydroxycholesterol on CFU-C proliferation and cholesterol biosynthesis are not solely a result of its inhibition of HMG CoA reductase, but are due in part to inhibition of enzymatic steps distal to mevalonic acid in the sterol synthetic pathway; and (4) mevalonic acid and cholesterol are independent requirements for CFU-C proliferation and differentiation in vitro.

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1–3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.

Collection of Pluripotential Hematopoietic Stem Cells by Cytapheresis

Successful complete hematopoietic reconstitution (CHR) using nonleukemic peripheral stem cells (PSC) after marrow ablation has been reported in animals but not man. Previous studies of cytapheresis products from humans, as a prelude to use for CHR, have documented the presence of committed myeloid (CFU-GM) and erythroid (BFU-E) precursors. We have examined mononuclear cell (MNC) products collected on the Fenwal CS3000 Blood Cell Separator for these plus the more primitive mixed (granulo-, erythro-, mono-, and megakaryocytic) cell colony-forming units (CFU-GEMM) and for various lymphocytic subpopulations (LSP). One to two-hour products contained 36 +/- 7 CFU-GEMM/10(6) MNC (mean +/- SE, n = 8) or 490 +/- 131/ml product. This compared favorably with blood (23 +/- .4/10(6) MNC or 46 +/- 8/ml, n = 14) and bone marrow (146 +/- 58/10(6) MNC, n = 12). Collection efficiency for E-rosette-positive cells approximated that for total lymphocytes and was variable for other LSP. Recovery of CFU-GEMM after freezing in 10% dimethylsulfoxide at a controlled rate and storage in liquid N2 was 54% +/- 8% (n = 8). Cytapheresis collection of large numbers of pluripotent hematopoietic precursors and demonstration of adequate recovery of these after cryopreservation, both previously unreported, are significant steps toward eventual CHR using nonleukemic PSC.

Collection of pluripotential hematopoietic stem cells by cytapheresis

Successful complete hematopoietic reconstitution (CHR) using nonleukemic peripheral stem cells (PSC) after marrow ablation has been reported in animals but not man. Previous studies of cytapheresis products from humans, as a prelude to use for CHR, have documented the presence of committed myeloid (CFU-GM) and erythroid (BFU-E) precursors. We have examined mononuclear cell (MNC) products collected on the Fenwal CS3000 Blood Cell Separator for these plus the more primitive mixed (granulo-, erythro-, mono-, and megakaryocytic) cell colony-forming units (CFU-GEMM) and for various lymphocytic subpopulations (LSP). One to two-hour products contained 36 +/- 7 CFU- GEMM/10(6) MNC (mean +/- SE, n = 8) or 490 +/- 131/ml product. This compared favorably with blood (23 +/- .4/10(6) MNC or 46 +/- 8/ml, n = 14) and bone marrow (146 +/- 58/10(6) MNC, n = 12). Collection efficiency for E-rosette-positive cells approximated that for total lymphocytes and was variable for other LSP. Recovery of CFU-GEMM after freezing in 10% dimethylsulfoxide at a controlled rate and storage in liquid N2 was 54% +/- 8% (n = 8). Cytapheresis collection of large numbers of pluripotent hematopoietic precursors and demonstration of adequate recovery of these after cryopreservation, both previously unreported, are significant steps toward eventual CHR using nonleukemic PSC.

Human Adherent Cell Contact-Mediated Modulation of Normal Myeloid Colony Formation<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

The effects of coincubation of normal nonadherent bone marrow cells on adherent monolayers created from human peripheral blood mononuclear cells or marrow cells were investigated. Nonadherent marrow cells were coincubated for 4 hours with peripheral blood adherent cells at ratios of adherent cells to marrow cells of 2:1 to 5:1. This coincubation suppressed subsequent neutrophilic agar colony growth but not eosinophilic growth. Further studies suggested that this suppression was a cell-cell-mediated process and not secondary to soluble factors. However, coincubation on marrow adherent cells caused increased neutrophilic colony recovery. The possible in vivo relevance is discussed.

Human megakaryocytes. II. Expression of platelet proteins in early marrow megakaryocytes.

Analysis of various platelet proteins by immunofluorescence demonstrated that platelet glycoproteins Ib, IIb, and IIIa, as well as plasma factor VIII antigen (factor VIII:AGN), platelet factor 4, and fibronectin are present in the vast majority of morphologically recognizable megakaryocytes. In addition, a small number of lymphoid-like mononuclear marrow cells, representing approximately 1.4--2.9/10(4) marrow cells, was found to express the same platelet proteins. This population of early marrow megakaryocytes is analogous to small acetylcholinesterase-positive rat and mouse marrow cells. Fc receptors for IgG were expressed in all megakaryocytes and megakaryocyte precursors, whereas the Ia antigen was detected only on a proportion of mature megakaryocytes and not on only early or precursor megakaryocytes. Platelet glycoproteins Ib, IIb, and IIIa, as well as factor VIII:AGN, and platelet factor 4 were established as distinct markers for marrow megakaryocytes and may be helpful for identifying megakaryocytic cells as well as for monitoring events of megakaryocyte differentiation.

Effect of oxygenated sterol compounds on human bone marrow granulocytic progenitor cells

Oxygenated sterol compounds are potent inhibitors of sterol and DNA synthesis in mammalian cells. We studied the effects of oxygenated sterols on human marrow granulocytic progenitor cells in vitro (CFU-C). 25-Hydroxycholesterol was found to be a potent inhibitor of sterol synthesis in marrow mononuclear cells, with 50% inhibition occurring at approximately 10(-7) M. This compound, as well as 6-ketocholestanol, 7- ketocholesterol, and 20 alpha-hydroxycholesterol, also demonstrated marked inhibition of CFU-C proliferation. The latter effect, which was not a result of direct cytoxicity of the compounds, was reversible by cholesterol, but not by mevalonic acid. We conclude that inhibition of sterol synthesis by oxygenated sterol compounds may be insufficient to explain their suppression of CFU-C proliferation.

Effect of Oxygenated Sterol Compounds on Human Bone Marrow Granulocytic Progenitor Cells

Oxygenated sterol compounds are potent inhibitors of sterol and DNA synthesis in mammalian cells. We studied the effects of oxygenated sterols on human marrow granulocytic progenitor cells in vitro (CFU-C)*. 25-Hydroxycholesterol was found to be a potent inhibitor of sterol synthesis in marrow mononuclear cells, with 50% inhibition occurring at approximately 10–7M. This compound, as well as 6-ketocholestanol, 7-ketocholesterol, and 20α-hydroxycholesterol, also demonstrated marked inhibition of CFU-C proliferation. The latter effect, which was not a result of direct cytoxicity of the compounds, was reversible by cholesterol, but not by mevalonic acid. We conclude that inhibition of sterol synthesis by oxygenated sterol compounds may be insufficient to explain their suppression of CFU-C proliferation.

Investigation of the Blastogenic and Cytotoxic Capabilities of Human Bone Marrow: Differences Between Aspirate and Rib Marrow

The use of marrow aspirates in bone marrow transplantation, clinical evaluation, and experimental studies has prompted the present study of the functional characteristics of mononuclear cells from aspirated (iliac crest) and curettaged (rib) marrow. In this manner, the relative contribution of peripheral blood contamination of aspirated bone marrow cells in investigating and understanding marrow function can be assessed. Human bone marrow specimens were collected using 3-5 ml aspirates from the iliac crest in 17 individuals and using curettage of ribs obtained from 13 individuals undergoing thoracotomy. The marrow samples were separated over discontinuous Ficoll-Hypaque gradients. Mononuclear cells from aspirated marrow had a high background stimulation and therefore no significant response to lectins (Con-A, E-PHA, PWM) or responsiveness in mixed lymphocyte culture (MLC), whereas mononuclear cells from rib marrow had a low unstimulated background activity and could be stimulated by E-PHA and allogeneic cells in MLC. Bone marrow cells obtained from aspirates and ribs were also studied as to their ability to mediate in vitro cellular cytotoxicity. Antibody-dependent cellular cytotoxicity (ADCC) and lectin-induced cytotoxicity (LICC) were observed using marrow mononuclear cells from both sources when red cells were employed as targets. In contrast, when cell line target cells were employed, rib mononuclear cells were not observed to mediate lysis, whereas mononuclear cells from aspirates did lyse these targets. The cytotoxicity observed with aspirates was therefore most likely due to peripheral blood contamination. In cell-mediated lympholysis (CML), rib mononuclear cells were not stimulated to kill allogeneic lymphocytes, while aspirated cells lysed allogeneic lymphocytes about 50% as well as did peripheral blood mononuclear cells (PBC), again, a reflection of peripheral blood contamination. We therefore conclude that pure bone marrow from humans, as assessed by studies using curettaged marrow from ribs, contains mononuclear cells capable of responding blastogenically to E-PHA and to cell surface alloantigens, as well as cells able to mediate LICC and ADCC against RBC, but does not include a population of cells able to lyse cell line targets in ADCC, spontaneous cell-mediated cytotoxicity (SCMC), or CML. These results indicate that the clinical evaluation, experimental study, and therapeutic use of bone marrow must be undertaken with the understanding that significant contamination with peripheral blood can occur when aspirates are used and that curettaged or biopsy material be used when true bone marrow is desired or needed.

Non-T, non-B acute lymphocytic leukemia cells enhance the mitogen induced proliferation of normal lymphocytes

Peripheral blood and bone marrow mononuclear cells (MNC) from patients with untreated non-T, non-B acute lymphocytic leukemia (ALL) were assessed for capacity to respond to the mitogens phytohemagglutinin (PHA), pokeweed (PWM) and staphylococcal enterotoxin A (SEA). At low mitogen concentrations considerable reactivity was observed to PHA and SEA, but not to PWM by patient MNC including specimens comprised of at least 90% leukemic cells. The responding cells were identified in cell separation experiments and by surface marker analysis as residual normal T-cells. The non-T, non-B leukemic cells enhanced the mitogen induced proliferation of responding lymphocytes as determined by co-culture experiments in which 5 × 10 3 or 10 4 MNC from normal donors were cultured with feeder layers of 3 × 10 5 mitomycin C treated MNC from autologous or unrelated normal donors or from ALL patients. In contrast to MNC feeder layers from normal donors, feeder layers of non-T, non-B leukemic cells enhanced normal lymphocyte reactivity to PHA and SEA but not to PWM as determined by 3H-thymidine uptake and cell survival. The potentiating effect of non-T, non-B leukemic cells was not mediated entirely by an allogeneic interaction as shown by the demonstration that frozen-thawed non-T, non-B leukemic cells enhanced the mitogen response of autologous MNC obtained during remission. Mitomycin treated feeder layers of leukemia cells from patients with acute myelogenous leukemia as well as bone marrow MNC from a normal donor or from lymphoma patients without bone marrow involvement failed to enhance PHA and SEA reactivity of normal lymphocytes. In addition, leukemia cells from patients with T-cell ALL did not potentiate normal lymphocyte reactivity in contrast to non-T, non-B ALL cells, providing a functional distinction between these two subtypes of ALL.

Erythropoietic enhancing activity (EEA) secreted by the human cell line, GCT.

Medium conditioned by the monocyte-like cell line GCT contains colony-stimulating activity (CSA), a mediator of in vitro granulopoiesis. Also, the conditioned medium (CM) contains erythroid-enhancing activity (EEA), which can be demonstrated in a system utilizing either nonadherent marrow or blood mononuclear cells, erythropoietin (1-2 units/ml), and 20 ml/dl fetal calf serum. Under these conditions, GCT CM enhances the growth of CFU-E and BFU-E. Attempts were made to characterize the molecular features of EEA. Serum-free GCT cell CM was fractionated on Sephacryl S200 and Ultrogel AcA54. EEA and CSA cochromatographed with apparent molecular weights of approximately 40,000 daltons on Sephacryl and approximately 30,000 daltons on Ultrogel. Fractionation on DEAE Sephacel led to an apparent separation of CSA from EEA; however, when diluted, the fractions containing CSA and EEA. Undiluted fractions containing potent CSA inhibited erythropoiesis; however, dilution of these fractions resulted in marked EEA. Diluted crude GCT CM and DEAE Sephacel fractions enriched in EEA were also capable of sustaining BFU-E in liquid culture and mediating erythropoietin-independent colony growth. CSA could not be unequivocally separated from EEA on concanavalin A-Sepharose, since the diluted void volume containing CSA also had EEA. EEA was present in CM boiled for 60 minutes, whereas CSA was markedly reduced but not abolished. The inverse relationship between CSA concentration and EEA mandates dilution of fractions when bioassayed for these two activities. Although CSA and EEA are similar in molecular weight, they appear to be partially separable by ion-exchange chromatography and heat stability.