Inhibition of human colony-forming-unit erythroid by tumor necrosis factor requires accessory cells.

Recombinant tumor necrosis factor (rTNF) inhibits erythropoiesis in vivo and in vitro. To study the mechanism of this inhibition, the effect of rTNF on highly purified human CFU-erythroid (E) (mean purity 63.5%), which were generated from peripheral blood burst-forming units-erythroid (BFU-E), was compared to its effect on unpurified human marrow CFU-E (mean purity 0.21%). Although growth of colonies from marrow CFU-E was inhibited by rTNF, no significant effect on purified BFU-E-derived CFU-E colony growth was found. Removal of accessory marrow cells by soy bean agglutinin (SBA) ablated the inhibition of marrow CFU-E colonies by rTNF. Inhibition of colony growth was then restored by adding back SBA+ cells, but not by adding T lymphocytes or adherent cells. Conditioned medium prepared from bone marrow mononuclear cells stimulated by rTNF inhibited the growth of colonies from highly purified BFU-E derived CFU-E resistant to direct inhibition by rTNF. These findings indicate that rTNF does not directly inhibit CFU-E, but requires accessory cells to decrease erythropoiesis. These accessory cells reside in the SBA+ cell fraction, but are neither T cells nor adherent cells. Therefore, in order to produce anemia, TNF must induce release or production of a factor that directly inhibits erythroid colony growth.

Human colony-forming units-erythroid do not require accessory cells, but do require direct interaction with insulin-like growth factor I and/or insulin for erythroid development.

The presence of heterogeneous erythroid progenitor cells, contaminant cells, or serum may alter erythroid colony development in vitro. To obtain highly purified colony-forming units-erythroid (CFU-E), we cultured partially purified human blood burst-forming units-erythroid (BFU-E) in methylcellulose with recombinant human erythropoietin (rHuEPO) for 7 d and generated cells that consisted of 30-60% CFU-E, but no BFU-E. A serum-free medium was used that allowed development of the same number of erythroid colonies as serum containing medium, but with a greater percentage of larger colonies. This medium consisted of delipidated crystalline bovine serum albumin, iron saturated transferrin, lipid suspension, fibrinogen, thrombin, Iscove's modified Dulbecco's medium/F-12[HAM], and insulin plus rHuEPO. When CFU-E were cultured in a limiting dilution assay and the percentage of nonresponder wells was plotted against cell concentration, both serum-free cultures and serum-containing cultures yielded overlapping straight lines through the origin indicating that CFU-E development did not depend on accessory cells and that insulin acted directly on the CFU-E. Human recombinant interleukin 3 (IL-3) and/or granulocyte-macrophage colony-stimulating factor had no effect on CFU-E growth, while they markedly enhanced BFU-E growth. Physiological concentrations of recombinant human insulin-like growth factor I (IGF-I) enhanced CFU-E growth in the absence of insulin and, together with rHuEPO in serum-free medium, provided a plating efficiency equal to that of serum-containing medium. Limiting dilution analysis in serum-free medium with IGF-I showed a straight line through the origin indicating that IGF-I also acted directly on the CFU-E and not through an effect on accessory cells. These data demonstrate that CFU-E do not require accessory cells, but do require IGF-I and/or insulin which act directly on the CFU-E.

Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.

Morphological and biochemical studies of human colony-forming units-erythroid (CFU-E) have been hindered by their extreme rarity. Since burst-forming units-erythroid (BFU-E) develop into CFU-E, we used normal human blood BFU-E to generate large numbers of highly purified CFU-E in vitro. Using density centrifugation, sheep erythrocyte rosetting, surface immunoglobulin-positive cell depletion, adherence to plastic, and negative panning with monoclonal antibodies, human blood BFU-E were purified from 0.017 to 0.368%, a 22-fold purification with a 43% yield. The panned cells were cultured in methylcellulose with recombinant erythropoietin (rEp) and conditioned medium for 9 d. These cells were then collected and CFU-E were further purified using adherence and density centrifugation. This yielded almost 10(7) erythroid colony forming cells with a purity of 70 +/- 18%. Analysis of these cells by light and electron microscopy showed 94% erythroid cells. The prominent cell was a primitive blast with high nuclear/cytoplasmic ratio, dispersed nuclear chromatin and a distinct large nucleolus. The relation between the number of erythroid colonies and the number of day 9 cells plated in plasma clots was a straight line through the origin with a maximum number of erythroid colonies at 1 U/ml of rEp and no erythroid colonies without rEp. Specific binding with 125I-rEp showed that 60% of the binding was inhibited by excess pure erythropoietin (Ep), but not by albumin, fetal calf serum, and a variety of growth factors or glycoproteins. By days 12-13 of cell culture, when the progenitor cells matured to late erythroblasts, specific binding markedly declined. In this study, human CFU-E have been isolated in sufficient purity to characterize the morphology of these rare cells and in sufficient numbers to measure specific binding of Ep.

Uptake and intracellular distribution of doxorubicin metabolites in B-lymphocytes of chronic lymphocytic leukemia.

The toxicity of doxorubicin metabolites was evaluated on lymphocytes of B-cell chronic lymphocytic leukemia. Only doxorubicinol was found to be cytotoxic for these lymphocytes, whereas exposure to aglycones at concentrations as high as 5 microM for 1 h had no effect on the proliferative capacity of these cells. After exposure of cells to isomolar concentrations of doxorubicin or its metabolites, uptake/retention of doxorubicinol was 23% of doxorubicin, and uptake/retention of aglycones was 5 to 13% of doxorubicin. Seventy to 90% of doxorubicin and 60 to 90% of doxorubicinol taken up/retained by the cells were detected in the cell nuclear fraction, whereas only 20 to 40% of the aglycones were localized in the cell nucleus. Cytotoxicity of metabolites was generally related to the proportion of drug taken up/retained by the cells and localized to the nuclei. The low uptake and nuclear localization may be at least partially responsible for the lack of cytotoxicity of aglycones on B-lymphocytes from chronic lymphocytic leukemia.

Quantitation of human megakaryocyte progenitors (CFU-M) in plasma clot culture by an indirect immunoperoxidase method.

We have developed an indirect immunoperoxidase method to detect human megakaryocytic colonies in plasma clot culture. A monoclonal antibody directed against platelet glycoprotein IIb/IIIa complex is incubated with plasma clots fixed to gelatin-coated slides with methanol. This antibody is subsequently linked to horseradish peroxidase by means of an avidin-biotin sandwich technique. Megakaryocytic colonies are identified by precipitation of benzidine by the horseradish peroxidase. This method detects growth of megakaryocytic colonies in culture, which is dependent on factors present in leukocyte-conditioned medium and is linear with respect to the concentration of human light-density nonadherent bone marrow cells plated at a concentration of 1.5-7 X 10(5) cells/ml. This method is simple to apply, uses objective criteria for recognition of megakaryocytes, and leaves cultures mounted and stained for permanent record.

Thrombopoiesis-stimulating factor: its effects on megakaryocyte colony formation in vitro and its relation to human granulocyte-macrophage colony-stimulating factor.

The effects of thrombopoiesis-stimulating factor (TSF) on human marrow megakaryocyte colony formation in vitro were studied by the plasma clot method. TSF was found to stimulate megakaryocyte as well as granulocyte-macrophage colony formation in vitro at optimal concentrations of 200-300 pg/ml of medium containing 2.5% horse serum. This colony-stimulating effect of TSF was not affected by polyclonal antibodies to human (h) interleukin 3 (IL-3) or to granulocyte colony-stimulating factor (G-CSF) but was neutralized by monoclonal or polyclonal antibodies to human granulocyte-macrophage colony-stimulating factor (hGM-CSF). In order to differentiate among cross-reactivity between TSF and hGM-CSF, induction of colony growth via release of GM-CSF, and presence of hGM-CSF in TSF preparations, TSF was tested on murine marrow cells, which are not responsive to hGM-CSF. TSF induced growth of murine megakaryocyte colony-forming units (CFU-MK) and granulocyte-macrophage colony-forming units (CFU-GM) in vitro with a dose response similar to that observed on human marrow cells; however, this effect could not be neutralized by antibodies to either human or murine GM-CSF. Using a double-antibody enzyme-linked immunosorbent assay, TSF preparations were found to contain 36 +/- 4 U of hGM-CSF per picogram of TSF protein. These findings indicate that hGM-CSF is responsible for the megakaryocyte colony-promoting effects of TSF on human marrow cells in vitro.

Replication and endoreplication in developing megakaryocytes in vitro.

To study the relation in time between replication and endoreplication and the relation between appearance of platelet-specific proteins and endoreplication in maturing megakaryocytes, peripheral blood mononuclear cells highly enriched in hematopoietic progenitors were cultured in liquid cultures and plasma clots in the presence of either interleukin-3 (IL-3) and stem cell factor (SCF) or medium conditioned by blood mononuclear cells stimulated by phytohemagglutinin (PHA). In plasma clots, megakaryocytic (MK) colonies appeared first on day 5 and reached a maximum by day 8, whereas the number of cells per colony increased until day 10, indicating that there was a single wave of MK colony formation. In liquid cultures, the first immunologically recognizable megakaryocytes appeared on day 5 and expressed GPIIb/IIIa and thrombospondin only, but all other platelet-specific protein markers appeared within 24 hours. Replating cells from liquid medium into plasma clots showed that 92 +/- 8% of day 6 GPIIb/IIIa-positive cells are capable of replicating. Their replicative potential decreased with age, however, so that between days 6 and 11, a linear correlation was noted between the logarithm of the percentage of megakaryocytes with replicative capacity and their age in culture. Replication ceased completely after day 10. In the presence of IL-3, polyploid megakaryocytes appeared at the same time that GPIIb/IIIa was expressed, and the megakaryocyte distribution into ploidy classes remained unchanged until day 20. In the presence of PHA-leukocyte conditioned medium (PHA-LCM), ploidy of megakaryocytes was shifted toward higher classes after day 6, and the process of endoreplication was completed by day 10. No changes in ploidy distribution were noted between days 10 and 20. These findings indicate that in the cohort of megakaryocytes derived from colony-forming units-megakaryocyte (CFU-MK), endoreplication can occur at an early stage of development, proceeds synchronously with replication, and is completed before the megakaryocytes exhaust their replicative potential.

Re-treatment of aplastic anemia with antithymocyte globulin or antilymphocyte serum.

Twenty-two patients with aplastic anemia were treated with antilymphocyte serum or antithymocyte globulin at Vanderbilt University and affiliated hospitals from 1980 to 1986. The median age was 42 (eight to 73 years); the male:female ratio was 8:14. Nineteen patients had severe aplastic anemia, and three had moderate disease. Twenty patients received antilymphocyte serum initially while two patients received antithymocyte globulin. Fifteen patients received fluoxymesterone 10 mg by mouth three times a day with antilymphocyte serum, and all received prednisone during the course of antilymphocyte serum or antithymocyte globulin. There were seven responses (31.8 percent) to the first course with four complete responses and three partial responses. Six of 15 patients who received fluoxymesterone showed a response, compared with zero of five treated without androgens (p less than 0.05). Eight patients with no initial response and a patient who experienced a relapse after a complete response were re-treated with either antithymocyte globulin (six) or antilymphocyte serum (three), with four of nine patients (44 percent) having a response (three complete responses, one partial response). Overall, 10 of 22 patients (45 percent) had a response (six complete responses, four partial responses). Median survival for those without a response is six months. Median survival for those with a response has not been reached, with follow-up ranging from 18 to 70 months. This study shows the benefit of a second cycle of antilymphocyte serum or antithymocyte globulin and a possible role for concomitant androgens in this treatment of aplastic anemia.

The pathogenesis of anemia in rheumatoid arthritis: a clinical and laboratory analysis.

Principal concepts concerning the anemia of RA are summarized in Tables 7 and 8. These concepts have been validated by our analysis of 93 anemic RA patients and by our review of the literature. The fact that anemia in RA may have one or more etiologies, occasionally in the same patient, mandates a reasoned approach to the analysis of anemia in every RA patient in whom it may occur. In particular, iron deficiency is common and determination of bone marrow iron content via an aspirate may be required for a definitive diagnosis. In those RA patients with anemia of chronic disease, the best therapy remains control of the underlying disease, most commonly with second line drugs and/or corticosteroids. The place for recombinant erythropoietin in the therapy of this anemia has not been defined; one specific role for erythropoietin may be in the preparation of RA patients for elective surgery, particularly hip arthroplasty, where correction of the anemia may either obviate the need for transfusion or may allow for donation of blood for purposes of autologous transfusion perioperatively. The pathogenesis of the anemia of chronic disease, as seen in RA anemia, is not completely understood. Inflammatory mediators, particularly the cytokines, appear to be important factors in the impairment of erythropoiesis. The mechanism by which these cytokines impair erythroid progenitor growth and hemoglobin production in developing erythrocytes is an important area for future study.

Deletion of the long arm of chromosome 5 (5q-) as a secondary event in the course of refractory anemia.

A 54-year-old chemical plant worker developed mild pancytopenia, with normal bone marrow morphology. Normal bone marrow cytogenetics were documented. The patient developed worsening anemia 5 years into his course. The bone marrow morphology remained normal, but four of 30 bone marrow metaphases examined showed deletion of the long arm of chromosome #5. Eight years into his course, the patient developed severe thrombocytopenia, and his bone marrow became hypercellular, with dysplastic changes. Deletion of the long arm of chromosome #5 was seen in all of 21 bone marrow metaphases examined. There had been no new exposure to potential mutagens during the course of the patient's illness. The occasional documentation of the late appearance of cytogenetic abnormalities during the course of clonal hematopoietic disorders implies that, in some cases at least, chromosomal abnormalities may not be primary pathogenetic events. The full expression of clonal disorders may require several pathogenetic events, which may occur in variable order.

Reversible hepatomegaly and diabetes mellitus in an adult with disseminated histiocytosis X.

Histiocytosis X rarely disseminates in an adult. The authors describe an unusual patients who presented with multiple areas of cutaneous and bone involvement. During the course of his disease he developed massive hepatomegaly. Aggregates of vacuolated histiocytes were found on liver biopsy. He subsequently developed diabetes mellitus complicated by ketoacidosis. Both his hepatomegaly and diabetes resolved spontaneously. No pancreatic nor pituitary abnormalities were identified. The combination of histiocytosis X, hepatomegaly, and diabetes mellitus has not been previously reported. The medical literature is reviewed with an emphasis on disseminated histiocytosis X in adults and the mechanism of glucose intolerance in liver disease.

Primary refractory anemia: clinical and laboratory study of erythropoiesis in 16 patients.

Erythropoiesis was studied in vitro in 16 selected patients with primary refractory anemia without excess of blasts who have been followed for an average of 4.8 years. The number of erythroid colonies and bursts grown in vitro from the patients' marrows did not correlate with any parameter of their disease or their prognosis. The response of marrow erythroid precursor cells to erythropoietin was found to be normal. In no case was a serum or IgG inhibitor of erythropoiesis detected either by quantitation of heme synthesized by marrow cells or by the erythroblast cytotoxicity assay. A clinically significant response of the anemia to corticosteroids was noted in three out of 14 patients. Ten patients died during the followup period, eight of them as a consequence of their hematologic disorder. Bone marrow aplasia with pancytopenia developed in six cases, increased number of marrow blasts in two cases, myelofibrosis with myeloid metaplasia in one case and a spontaneous remission in another case. Refractory anemia without excess of blasts is a heterogeneous disorder with variable natural history including evolution into marrow aplasia.

Case report: granulocyte colony-stimulating factor overcomes severe neutropenia of large granular lymphocytosis.

Large granular lymphocytosis (LGL) is characterized by enhanced proliferation of T lymphocytes that have antibody-dependent cell-mediated cytotoxicity or natural killer cell activity and that often produce severe cytopenias, including neutropenia. When a 68-year-old man with seropositive rheumatoid arthritis and severe neutropenia was examined, he was found to have LGL with a T cell gene rearrangement, indicating the presence of a clonal population of T lymphocytes. The patient was admitted with a fever of 102 degrees F and a nonhealing ulcer over the right tibia. When the infection did not respond to intravenous antibiotics, granulocyte colony-stimulating factor (GCSF) therapy was started at 5 micrograms/kg subcutaneously each day. The neutrophil count promptly increased and the patient subsequently defervesced and was able to have a skin graft placed, which healed without difficulty. GCSF, which is known to be an effective therapeutic agent for neutropenia associated with chemotherapy and bone marrow transplantation, also was a very valuable treatment for the life-threatening neutropenia of LGL.

Treatment of the anemia of predialysis patients with recombinant human erythropoietin: a randomized, placebo-controlled trial.

Recombinant human erythropoietin (r-HuEPO) was administered in two phases to 12 patients with chronic renal insufficiency (creatinine clearances of 0.17-0.51 ml/second [10-30 ml/minute]) and uremic anemia. In addition to the routine tests done as part of a multicenter clinical trial, our patients had serial red cell mass measurements, quantitation of bone marrow stem cells, and marrow cytogenetic analysis. During the first eight weeks (acute phase), an equal number of patients was randomized to placebo or one of three doses of r-HuEPO (50, 100 or 150 unit/kg intravenously three times weekly). All three patients receiving 150 unit/kg responded by increasing their packed cell volume (PCV) to the normal range within eight weeks. There were lesser responses in PCV at the two lower doses of r-HuEPO and no response in the placebo group. The 51Cr red cell mass also increased significantly in a dose-related manner in patients receiving r-HuEPO but did not change in the placebo group. Marrow studies revealed increases in erythroid, megakaryocyte, and granulocyte-monocyte progenitor cells in those patients on r-HuEPO, but no mutagenic effects were seen. Subsequently, ten patients received open label r-HuEPO. During this maintenance phase, all ten achieved or maintained a normal PCV. Several adverse events occurred, but none were definitely linked to r-HuEPO. Recombinant human erythropoietin is an effective and potent treatment of anemia caused by renal failure.

Aplastic anemia and pure red cell aplasia.

The role of known hematopoietic growth factors in the pathogenesis of aplastic anemia and congenital hypoplastic anemia has been extensively studied and no evidence has been obtained that deficiency of these factors contributes to the hypoproliferative state in these disorders. Clonal hematopoiesis seems to be present at least in a small percentage of cases of aplastic anemia, a finding that needs further investigation. Androgens were shown to be beneficial only for women with aplastic anemia treated with antilymphocyte globulin. Unrelated-donor bone marrow transplantation is becoming a realistic approach for children and very young adults with aplastic anemia, but in older groups the survival is very poor. New observations on abnormalities of lymphokines and cytokines in Fanconi's anemia have been described, but their pathogenetic significance remains unknown. A large number of studies have excluded the possibility that abnormalities of c-kit/SCF genes and their expression are responsible for the erythroid aplasia in Diamond-Blackfan syndrome. Cyclosporine was found to be an effective treatment for pure red cell aplasia associated with chronic lymphocytic leukemia. The cell membrane receptor for B19 parvovirus has been identified as the P antigen. Long-term studies showed that in 20% of patients with homozygous sickle cell disease, infection by B19 does not cause erythroid aplasia.

Effects of recombinant erythropoietin on murine megakaryocytic colony formation in vitro.

The availability of pure recombinant erythropoietin permits the study of its effects on hematopoietic progenitors free from those of other factors that may copurify with it. Mouse bone marrow cells were cultured in plasma clots in the presence of recombinant human erythropoietin. This factor supported megakaryocytic colony formation in a dose-dependent fashion, with plateau growth at 1 U erythropoietin per milliliter of culture medium. Erythropoietin did not increase the number of granulocyte-macrophage colonies. Plasma clots per se were not essential for megakaryocytic colony formation, because recombinant erythropoietin also supported colony growth in soft agar containing 25% serum. However, little colony formation was observed in serum-free soft agar cultures containing erythropoietin. Colony formation supported by suboptimal concentrations of erythropoietin was additive to that supported by suboptimal amounts of medium conditioned by pokeweed mitogen-stimulated spleen cells or by WEHI-3 cells. Delayed addition of conditioned medium to cultures resulted in a 50% to 100% decline in the number of megakaryocytic colonies by 12 to 48 hours, which was abolished by the inclusion of erythropoietin in the original culture medium. Delayed addition of erythropoietin by 24 hours to cultures resulted in loss of its effect on colony formation. These results indicate that erythropoietin has an effect on murine megakaryocytic colony formation in vitro and show that at least a portion of this effect is exerted during the early stages of colony development.

Antibody-dependent cellular cytotoxicity to allogeneic but not autologous erythroblasts in vitro.

Antibody-dependent cellular cytotoxicity (ADCC) is a very sensitive mechanism for immune injury of target cells, which utilizes extremely low concentrations of antibody. We have developed a method for demonstrating this type of cytotoxicity to normal human erythroblasts. The latter were enriched 3- to 4-fold and were then labeled with 59Fe. Blood lymphocytes from the same donor were enriched to 93% and were added as effector cells at a 60:1 ratio to the target cells. After 4 hr at 37 degrees C, as 5- to 10-fold increase in the release of 59Fe occurred when the plasma or IgG from patients with pure red-cell aplasia was present. This activity was not present when the effector cells were absent. However, this activity was found in the remission plasmas of patients and was not found when autologous erythroblasts were used. These studies demonstrate a method for detecting ADCC to allogeneic normal human erythroblasts. This ADCC does not appear to be related to the disease since a similar autoimmune activity to the patients' own erythroid cells was not detected. Further studies are suggested using other effector cells in this system.

Effect of pure erythropoietin on DNA-synthesis by human marrow day 15 erythroid burst forming units in short-term liquid culture.

The effect of pure erythropoietin (EP) on human marrow day 15 burst-forming units-erythroid (BFU-E) was studied using a short-term liquid culture system containing 30% human serum. Non-adherent marrow cells were cultured in liquid medium for 0-48 h and then the number of BFU-E was assayed by the use of the plasma clot method. The addition of 1 U/ml of EP into the liquid culture medium resulted in maintenance of the number of BFU-E assayed after 24-48 h of incubation. The number of BFU-E recovered after 24-48 h culture was directly proportional to the concentration of EP present in the liquid medium. In addition, the proliferative status of BFU-E before and after exposure to EP was studied by 3H-thymidine and hydroxyurea suicide. It was found that EP doubles the percentage of BFU-E in DNA synthesis after 24-48 h of incubation in the liquid medium. This effect of EP on DNA synthesis by bone marrow day 15 BFU-E is detectable as early as 6 h after the onset of incubation and at EP concentrations as low as 0.2 U/ml of medium, a concentration present in the serum of moderately anaemic patients. The human marrow day 15 BFU-E is an EP-responsive cell and pure EP can induce it into DNA synthesis.