The influence of steroid hormone metabolites on the in vitro development of erythroid colonies derived from human bone marrow.

Certain C19 and C21 steroid metabolites, when incubated with normal human bone marrow cells in culture, increased the number of erythroid colonies in the presence of erythropoietin. Among a number of pairs of C5 epimeric steroids tested, most 5beta (A:B cis) steroids stimulated the growth of both early erythroid progenitor cells (BFU-E) and late erythroid progenitor cells (CFU-E), whereas only a few 5alpha-(A:B trans) steroids stimulated the growth of CFU-E. No 5alpha-compounds of six pairs of steroids studied were found to stimulate BFU-E formation. This structure-activity relationship conforms with that previously observed in studies of steroid induction of ALA-synthase in avian embryo liver cells and hemoglobin synthesis in the cultured avian blastoderm. When human bone marrow cells were preincubated with the steroids for 2 d, followed by incubation with erythropoietin, only the 5 beta-compounds stimulated the growth of BFU-E. Similarly, when addition of steroids was delayed in relation to erythropoietin in the culture, only the 5 beta-derivative of a pair of C5 epimeric compounds displayed an enhancing effect on the growth of BFU-E. This effect required that the steroid addition be made no later than 48 h after initiation of the culture. These data demonstrate that certain natural steroid metabolites significantly stimulate erythropoiesis in normal human bone marrow cells in culture. They also indicate that 5 beta-compounds are more stimulatory than their 5 alpha-epimers, and they suggest that these 5 beta-steroids act preferentially on very primitive erythroid progenitor cells, probably on BFU-E.

Increased glucocorticoid receptor concentration in macrophage differentiation of myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate.

HL60 cells, human promyelocytic leukemia cells, can be induced to differentiate into more mature myeloid forms by dimethyl sulfoxide (DMSO) or retinoic acid (RA) and into macrophage-like cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) or related compounds. Macrophage differentiation of HL60 cells by TPA treatment induced a 3- to 4-fold increase in glucocorticoid receptor concentration per cell and a 2- to 3-fold increase in glucocorticoid receptor concentration per mg of protein. The ability of TPA derivatives to increase glucocorticoid receptor concentration paralleled their ability to induce macrophage differentiation. Macrophage differentiation of other myeloid leukemia cells by TPA treatment induced a 2- to 3-fold increase in glucocorticoid receptor concentration per cell. Exposure of T-lymphoblasts or erythroleukemia cells to TPA did not affect glucocorticoid receptor concentration. Myeloid differentiation of myeloid leukemia cells by DMSO or RA induced no significant change in glucocorticoid receptor concentration. The increase in glucocorticoid receptor concentration in macrophage differentiation of myeloid leukemia cells with TPA was considered to depend, not on TPA treatment, but on the process of macrophage differentiation. Further, glucocorticoid receptor concentration can be a sensitive marker of macrophage differentiation of myeloid leukemia cells.

Induction of the receptor for erythropoietin in murine erythroleukemia cells after dimethyl sulfoxide treatment.

Biologically active 125I-labeled human recombinant erythropoietin (EPO) was used to demonstrate specific receptors for this erythroid-specific hemopoietic growth factor on the cell surface of murine erythroleukemia cell clone B8. The binding of radioiodinated EPO to these cells was time and temperature dependent, specific, saturable, and reversible. During erythroid differentiation by dimethyl sulfoxide, B8 cells displayed a rapid and marked increase in the amount of specific 125I-EPO binding before the appearance of hemoglobin-containing cells. Scatchard analysis of the saturation binding data revealed that B8 cells had a single class and low number (350 to 650) of EPO receptors per cell with an apparent Kd of 1.2 to 1.4 nM. In addition, the number of EPO receptors on B8 cells was increased twice by induction with DMSO for 1 day, but the binding affinity of EPO toward its receptors did not change significantly. Affinity cross-linking experiments with disuccinimidyl suberate demonstrated two radiolabeled components with apparent molecular weights of 145,000 and 130,000 under both reducing and nonreducing conditions. Labeling of the two components was inhibited by incubation of cells with unlabeled EPO. These results suggest that some murine erythroleukemia cells potentially express EPO receptors as a differentiation marker of erythroid lineage, which contain two polypeptides with molecular weights of 109,000 and 94,000.

Elimination of small cell lung cancer cells in vitro from human bone marrow by a monoclonal antibody.

We report here a useful method for elimination of small cell lung cancer cells in vitro from bone marrow. A monoclonal antibody, TFS-2, which mediates complement lysis and recognizes an antigen present on small cell lung cancer cells but not lymphoid cells or bone marrow cells, was used to clear infiltrated bone marrow. The antibody in the presence of complement effectively killed tumor cells, but it was not cytotoxic to bone marrow cells. When mixed populations consisting of tumor cells and bone marrow cells were treated with antibody and complement, the tumor cells were also effectively killed, except when large numbers of bone marrow cells were present, whereas TFS-2 had no significant effect on bone marrow stem cells, as judged by colony-forming unit assays.

Effects of 12-O-tetradecanoylphorbol-13-acetate on the proliferation and differentiation of normal and leukemic myeloid progenitor cells.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on myeloid colony formation were investigated by initial and delayed addition of TPA to the cultures. In the human placental conditioned medium-stimulated cultures, TPA inhibited normal myeloid colony formation without any change in colony morphology when added at the beginning of culture. However, macrophage-like transformation of myeloid colonies by TPA was clearly observed after the delayed addition of TPA. Colonies and clusters already formed at the time of TPA addition wee exclusively neutrophilic. Two days after TPA addition, many colonies apparently contained macrophage-like cells. Within 4 days after TPA addition, almost all myeloid colonies transformed into the macrophage type. Parallel study of initial and delayed addition of TPA revealed that this macrophage-like transformation of neutrophilic colonies occurred at high concentrations of TPA that would fully inhibit colony formation if added initially. TPA caused similar effects on leukemic colony formation.

Characterization and molecular features of the cell surface receptor for human granulocyte-macrophage colony-stimulating factor.

The receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF) on the surfaces of normal and leukemic myeloid cells were characterized using 125I-labeled bacterially synthesized GM-CSF. The binding was rapid, specific, time dependent, and saturable. Scatchard analysis of the 125I-GM-CSF binding to peripheral blood neutrophils indicated the presence of a single class of binding site (Kd = 99 +/- 21 pM; 2,304 +/- 953 sites/cell). However, for peripheral blood monocytes and two GM-CSF-responsive myeloid cell lines (U-937 and TF-1), the Scatchard plots were biphasic curvilinear, which were best fit by curves derived from two binding site model: one with high affinity (Kd1 = 10-40 pM) and the other with low affinity (Kd2 = 0.9-2.0 nM). For U-937 cells, the number of high-affinity receptors was 1,058 +/- 402 sites/cell and that of low-affinity receptors was estimated to be 10,834 +/- 2,396 sites/cell. Cross-linking studies yielded three major bands with molecular masses of 150 kDa, 115 kDa, and 95 kDa, which were displaced by an excess amount of unlabeled GM-CSF, suggesting 135-kDa, 100-kDa, and 80-kDa species for the individual components of the human GM-CSF receptor. These bands comigrated for different cell types including peripheral blood neutrophils, U-937 cells and TF-1 cells. In experiments using U-937 cells, only the latter two bands appeared to be labeled in a dose-dependent manner in a low-affinity state. These results suggest that the human GM-CSF receptor possibly forms a multichain complex.

Laboratory findings and clinical courses of 33 patients with granular lymphocyte-proliferative disorders.

The hematological and immunological findings and clinical courses of 33 patients (13 male, 20 female; median age at presentation, 60 years) with granular lymphocyte-proliferative disorders (GLPD) are presented. Based on the surface phenotypes of peripheral blood granular lymphocytes (GL), the GLPD were divided into CD3+ T cell-lineage GLPD (T-GLPD) and CD3- CD16+ natural killer (NK) cell-lineage GLPD (NK-GLPD). Twenty-one patients had T-GLPD, and 12 had NK-GLPD. One patient with T-GLPD and two patients with NK-GLPD had progressive clinical courses and died of the disease despite receiving combination chemotherapy. Twelve patients with T-GLPD were found to have severe anemia at presentation or during the course of the disease; four of them fulfilled the diagnostic criteria of pure red cell aplasia, and the others had closely related conditions. Six of these 12 patients were treated with cyclophosphamide, and all responded to the treatment. In 16 patients, the clinical course was stable, and spontaneous regression was observed in two patients. Since some of the patients with NK-GLPD had stable clinical courses while some had progressive clinical courses, clinical findings in these two groups were compared. We found, taking into consideration our cases and those reviewed in the literature, that age less than 40 years, fever, lymph node swelling, hepatosplenomegaly, and GL with CD16(Leu-11)-CD56+CD57- phenotype and low or absent antibody-dependent cellular cytotoxicity seemed to be predictors of a progressive clinical course.

Pure erythropoietic colony and burst formations in serum-free culture and their enhancement by insulin-like growth factor I.

Recombinant human insulin-like growth factor I (IGF-I) increased human and murine erythropoietic colony formation in serum-free culture. In order to investigate the effects of purified factors such as IGF-I on hemopoietic progenitor cells, we have established a serum-free culture system which supports the clonal growth of CFU-E- and BFU-E-derived colonies. Exogenously supplied ingredients were bovine serum albumin (BSA), transferrin, lipid suspensions, 2-mercaptoethanol, and recombinant human erythropoietin (epo). Among these, BSA and cholesterol were found to be essential ingredients. The optimum concentration of BSA sufficient to grow BFU-E was 3%. Erythroid colony and burst formation of human and murine marrow cells was enhanced twofold (p less than 0.05) by a physiological concentration of recombinant human IGF-I. Potentiation was observed in a dose-dependent manner between 10(-9) and 10(-7) M. A few murine CFU-E colonies were formed in the absence of epo. These results suggest that IGF-I has a supportive effect on the proliferation and differentiation of erythroid precursor cells stimulated by epo and that its action is synergistic with that of epo.

Partial prevention of compactin (ML-236B) inhibition of in vitro hematopoiesis by dolichol and dolichyl phosphate.

We have reported that the exogenous addition of dolichyl phosphate (Dol-P) enhances the colony-forming capacities of early erythroid progenitors (BFU-E), late erythroid progenitors (CFU-E), and granulocyte-macrophage progenitors (CFU-GM) in adult mouse bone marrow, and that dolichol (Dol) enhances that of only CFU-E (Int. J. Cell Cloning 3:313, 1985). Compactin (2.5-10 microM), a specific inhibitor of mevalonate biosynthesis that causes a decrease of endogenous Dol biosynthesis, inhibited colony formation of CFU-GM. Exogenous addition of Dol-P partially prevented this inhibition, but Dol and the other mevalonate metabolites, such as cholesterol, coenzyme Q10, and isopentenyladenine, could not. In addition, we have found that the colony-forming capacity of CFU-E in fetal mouse liver was not enhanced by exogenous Dol or Dol-P. But the decrease of colony formation or DNA synthesis of fetal CFU-E in the presence of compactin was prevented by the exogenous addition of Dol or Dol-P.

Enhancement of human T lymphocyte colony formation by 12-O-tetradecanoylphorbol-13-acetate (TPA).

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocyte lymphocyte colony formation in vitro were investigated. The number of T lymphocyte colonies was increased 4-5 times over that of controls by the addition of TPA (10(-7) - 10(-9) M) to phytohemagglutinin (PHA)-containing cultures. Few colonies were observed when stimulated with TPA in the absence of PHA. In the cultures containing a sufficient amount of exogenous T cell growth factor (TCGF), the enhancement of T lymphocyte colony formation by TPA was not observed. TPA enhanced TCGF production by peripheral lymphocytes stimulated with PHA. The optimal concentrations of TPA for T lymphocyte colony formation were similar to those for TCGF production. These findings suggest that TPA enhanced T lymphocyte colony formation by stimulating endogenous TCGF production. Interestingly, T lymphocyte colony formation was not inhibited even at high concentrations of TPA that usually inhibit myeloid and erythroid colony formation. This difference may be due to different sensitivities to TPA between T lymphocyte colony-forming cells and myeloid and erythroid colony-forming cells.

Recombinant immune interferon inhibits leukemic cell growth by a monocyte-macrophage-mediated mechanism.

We have investigated direct and monocyte-macrophage (Mono/M phi)-mediated indirect effects of recombinant immune interferon (IFN-gamma) on the growth of established leukemic cell lines (K562, KG1, ML1, HL60, U937, and THP1). The direct antiproliferative effects of IFN-gamma on these leukemic cells were mild or negligible, when estimated by 3H-thymidine incorporation. Indirect effects were assessed by the growth pattern of leukemic cells cocultured with Mono/M phi that were pretreated with INF-gamma. While the leukemic cell growth was slightly suppressed by untreated Mono/M phi, this suppression was significantly augmented by the treatment of Mono/M phi with IFN-gamma (10-10,000 U/ml). In addition, the indirect effects of IFN-gamma on leukemic cell growth were examined at different stages of maturation of Mono/M phi. The augmentation of cytotoxicity was detected only when mature Mono/M phi were treated with INF-gamma. This suggests that IFN-gamma acts on tissue macrophages and augments their cytotoxicity against leukemic cells.

Synergistic effect of dolichyl phosphate and human recombinant granulocyte colony-stimulating factor on recovery from neutropenia in mice treated with anti-cancer drugs.

Severe hematopoietic injury in mice was induced by using either 5-fluorouracil, adriamycin, mitomycin C, or vinblastine. Daily subcutaneous administration of purified human recombinant granulocyte colony-stimulating factor (rG-CSF; 0.3-10.0 micrograms/day) markedly accelerated recovery from the drug-induced granulocytopenia in a dose-dependent manner, as reported previously. On the other hand, daily intraperitoneal administration of dolichyl phosphate (Dol-P) also enhanced granulopoiesis to accelerate recovery from granulocytopenia, although the effect of Dol-P was relatively moderate as compared with that of rG-CSF. A synergistic recovery of granulopoiesis was observed when Dol-P was administered together with rG-CSF to the mice treated with anti-cancer drugs. Joint use of Dol-P (1 mg/day) and rG-CSF (0.3 micrograms/day) was as effective as a higher dose of rG-CSF (3 micrograms/day). Joint use of Dol-P (1 mg/day) and rG-CSF (3 micrograms/day) was sometimes more effective.

Binding of erythropoietin to CFU-E derived from fetal mouse liver cells.

The binding of recombinant erythropoietin (EPO) to fetal mouse liver cells (FMLC) was investigated using a radioiodinated derivative which retained full biological activity. FMLC were fractionated using a preformed Percoll density gradient. Using the fractionated FMLC, the ability to form CFU-E colonies in a semisolid culture was examined, and the binding of [125I]EPO was measured. The highest specific binding of [125I]EPO was observed in a fraction with a density between 1.062 and 1.076 g/ml. The same fraction showed the highest ability to form CFU-E-derived colonies. After suspension culture of FMLC with EPO for 2 days, differentiated erythroid cells with higher density markedly increased. The specific binding of [125I]EPO to these cells almost disappeared with differentiation. Scatchard analysis with cells of the CFU-E-enriched fraction showed a nonlinear curve, suggesting the existence of two classes of binding sites. One binding site was high-affinity (Kd1 = 0.41 nM), and the other low-affinity (Kd2 = 3.13 nM). These results suggest that the expression of EPO receptors on the erythroid cells is highest in CFU-E.

Binding properties and proliferative potency of insulin-like growth factor I in fetal mouse liver cells.

Specific high-affinity receptor(s) for insulin-like growth factor I have been identified in fetal mouse liver cells (FMLC) rich in late erythroid progenitors (CFU-E). Competition for [125I]IGF-I binding by IGFs and insulin demonstrated the presence of Type-I IGF receptors. Scatchard analysis of the binding data revealed a single class of receptors (Kd, 1.2 nM; R0, 600 sites per cell). Erythroid colony formation and DNA synthesis by these cells were enhanced by IGF-I alone or in combination with erythropoietin (Epo). Subfractionations of FMLC using Percoll density gradients showed that a significant part of [125I]IGF-I binding was observed in the CFU-E-enriched fraction and that the erythroid colony formation was mostly enhanced by IGF-I in the same fraction. IGF-I stimulated the phosphorylation of the beta-subunit of the Type-I receptors. These results indicate that IGF-I modulates the Epo-stimulated proliferation and differentiation of erythroid progenitors via its specific receptors.

Binding of iodinated erythropoietin to rat bone marrow cells under normal and anemic conditions.

Specific binding sites for erythropoietin (Epo) were shown in normal and anemic rat bone marrow cells using [125I]labeled human recombinant Epo. When rats were treated once or several times with phenylhydrazine or malotilate, or by phlebotomy, the serum Epo level determined by RIA began to increase rapidly. Thereafter, both the number of erythroid colony-forming unit (CFU-E)-derived colonies and the Epo binding capacity of bone marrow cells increased almost simultaneously in response to induced anemic states, suggesting that the amount of Epo binding in bone marrow cells may reflect in vivo erythropoiesis. Scatchard analysis of the binding data from normal rats revealed the presence of a single class of binding sites (Kd = 0.18 +/- 0.04 nM, 38 +/- 5 sites/cell). In anemic states, the apparent average receptor number per cell increased (52-62 sites/cell) without changing in binding affinity toward Epo. Furthermore, [125I]Epo was cross-linked to the cell surface molecule of approximately 165 kd in nonreducing conditions and 75 kd in reducing conditions. Autoradiographic analysis indicated that Epo receptors were distributed on immature erythroid cells. Proerythroblasts were the most heavily labeled, whereas orthochromatic erythroblasts and cells of myeloid and lymphoid lineages were not labeled. Calculations based on Scatchard and autoradiographic analysis showed that proerythroblasts have 390 receptor sites per cell, twice as many as basophilic or polychromatophilic erythroblasts have. These results are consistent with the stage-specific action of Epo in physiological differentiation of erythroid cells.

A new method for permanent preparations of hemopoietic cells cultured in methylcellulose medium.

To obtain permanent preparations for morphologic examination of hemopoietic cells cultured in methylcellulose medium, a new method designated as "the membrane filtration technique" has been devised. Permanent preparations of erythroid colonies, bursts and leukemic colonies, which have been grown mainly in methylcellulose medium, are illustrated in this paper. The stages of differentiation of cells within these colonies can be easily recognized by means of Wright-Giemsa staining.

Increased neutrophil respiratory burst in myeloproliferative disorders: selective enhancement of superoxide release triggered by receptor-mediated agonists and low responsiveness to in vitro cytokine stimulation.

The neutrophil superoxide (O2-)-producing capacity in 57 patients with chronic myeloproliferative disorders (MPDs) and eight patients with chronic myelomonocytic leukemia (CMML) was investigated. O2- release in neutrophils stimulated by chemotactic peptide was markedly increased in all types of chronic MPD, including chronic myelogenous leukemia in both chronic phase and blastic crisis, polycythemia vera, and essential thrombocythemia, but was normal in CMML, which is thought to be a myelodysplastic disorder rather than MPD. Increase in O2(-)-producing capacity in MPD was also observed when other receptor-mediated agonists such as interleukin-8 and concanavalin A were used, but not when phorbol ester, a direct activator of protein kinase C, was used as the triggering agonist of O2- release. Priming effects of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and tumor necrosis factor (TNF) on chemotactic peptide-induced O2- release was observed in all patients with MPD and CMML, though fold enhancement of priming effects was much less in MPD compared with normal subjects. In addition, the priming effects of TNF were less than those of GM-CSF in 10 cases, whereas the priming effects of TNF were consistently and markedly greater than those of GM-CSF in normal subjects. Tyrosine phosphorylation of 42-kDa protein stimulated by G-CSF, GM-CSF, and TNF was observed in CML neutrophils to be identical to that in normal neutrophils. Present results indicate specific potentiation of the receptor-mediated route of signaling that is linked to the respiratory burst and downregulated responsiveness to cytokines in neutrophils in patients with all types of chronic MPD, suggesting in vivo priming of patient neutrophils via certain mechanism by cytokines or related stimuli in these hematological disorders.

Cyclosporin A inhibits directly in vivo keratinocyte proliferation of living human skin.

A direct in vivo antiproliferative effect of cyclosporin A (CsA) on human epidermal keratinocytes (EK) grafted onto nude mice was evaluated. Using pulse-labeling of 5-bromo-2'-deoxyuridine (BrdU), a thymidine analogue incorporated into the nuclei of DNA-synthesizing (S-phase) cells, the antiproliferative effect of CsA was revealed as a decrease in the number of BrdU-positive human EK grafted onto nude mice receiving a daily subcutaneous injection of 50 mg/kg of CsA. The blood level of CsA in the treated mice, evaluated by a radioimmunologic assay, was 679 +/- 501 ng/ml (n = 3). Using an antibody to leukocyte common antigen, it was shown that no human lymphocytes were present in the grafted skin. Therefore, this antiproliferative effect of CsA on human EK seems to be due to a direct effect on EK rather than to lymphocyte regulation.

Clinical features of the neutropenic host: definitions and initial evaluation.

Initial evaluation of patients with febrile neutropenia includes a thorough history and physical examination; a complete blood cell count; measurement of serum creatinine, blood urea nitrogen, transaminases, and C-reactive protein; and culture of blood (samples from a peripheral vein and/or catheter). Chest radiography is indicated for patients with respiratory signs or symptoms. Signs and symptoms of inflammation may be minimal or absent. However, a search should be undertaken in the sites most commonly infected, including the periodontium, pharynx, lower esophagus, lung, perineum, eyes, and skin. Blood samples, including samples from catheter lumen(s), if present, and a peripheral vein, should be obtained for cultures for bacteria and fungi. Urine culture is indicated in the presence of signs or symptoms of urinary tract infection, a urinary catheter in place, or abnormal results of urinalysis. Fever is defined as a single axillary temperature measurement of > or =37.5 degrees C (oral temperature of > or =38.0 degrees C). Neutropenia is defined as a neutrophil count of <1000 cells/mm3.

Randomized trial of cefepime monotherapy or cefepime in combination with amikacin as empirical therapy for febrile neutropenia.

A multicenter open randomized trial was conducted to compare cefepime monotherapy with cefepime/amikacin combination (dual) therapy in treating febrile neutropenic patients with hematologic disorders. Among the 189 evaluable patients, 5.8% had microbiologically and 10.6% had clinically documented infections. Excellent response was seen in 32.6% and 45.7% of monotherapy and dual therapy recipients, respectively, at day 3 (P=.065). At day 3, patients with neutrophil counts of <500/ mu L receiving dual therapy had a better response than did those receiving monotherapy (45% vs. 27.6%; P=.024). The same was true for patients with leukemia. Adverse events were minimal, and early death was observed in 7 patients in the dual therapy group and 5 patients in the monotherapy group. Overall, cefepime monotherapy is as effective as dual therapy for the initial treatment of febrile neutropenic patients. Further study is warranted for patients with severe neutropenia and leukemia who may benefit from dual therapy.