Cyclic hematopoiesis: effects of lithium on colony-forming cells and colony-stimulating activity in grey collie dogs

The cycling of blood cell counts in grey collie dogs with cyclic hematopoiesis can be eliminated by treatment with oral lithium carbonate. To explore the mechanism by which lithium alters this stem cell disorder, studies of bone marrow granulocyte-macrophage progenitor cells (CFU-C), neutrophil colony-forming cells (neutrophilic CFU-C), and colony-stimulating activity (CSA) were performed. In untreated dogs, the proportions of CFU-C were found to fluctuate cyclically, but the cyclic fluctuations in neutrophil colony-forming cells were even more marked, with numbers decreasing to undetectable levels during each period of neutrophilia. Dogs on lithium, however, did not cycle the numbers of total or neutrophilic CFU-C. Tritiated thymidine suicide rates were not altered by treatment with lithium. Serum CSA levels and bone marrow cell elaboration of CSA were not increased by lithium. These studies suggest that lithium corrects cyclic neutropenia by a direct effect on the differentiation and proliferation of CFU-C; normalization of the proportion of CFU-C that enter neutrophilopoiesis appears to be an important effect of the lithium therapy.     

Canine cyclic haematopoiesis: the effect of endotoxin on erythropoiesis

We have examined the effects of chronic endotoxin treatment on erythropoiesis in six grey collies with cyclic haematopoiesis. Blood reticulocytes, bone marrow erythroid colony (EC) forming cells, serum iron and erythropoietin (ESF) values showed regular periodic fluctuations in untreated grey collies. Daily endotoxin injections eliminated the cyclic fluctuations of reticulocytes and EC. The mean serum iron values were increased and recurrent hypoferraemia eliminated, while the mean serum ESF values were reduced. The cyclic fluctuations of serum ESF values were no longer apparent in the endotoxin treated grey collies. Tritiated thymidine suicide of the marrow EC forming cells failed to show cyclic changes either in untreated or endotoxin treated dogs. The ESF sensitivity of EC in the grey collie was unchanged during endotoxin treatment and was not different from normal dogs. Endotoxin appears to alter periodic erythropoiesis by stabilizing the flux of cells into the committed erythroid precursor cell pool from a more primitive stem cell compartment.     

Lithium therapy of canine cyclic hematopoiesis

Treatment of cyclic hematopoiesis in the grey collie dog with lithium carbonate eliminated the recurrent neutropenia and normalized the other blood cell counts. These findings suggest that human cyclic hematopoiesis may be successfully treated with lithium. The effects of lithium on the monocytes, platelets, and reticulocytes, as well as the neutrophils, suggest that lithium operates on basic regulatory mechanisms affecting the most primitive hematopoietic precursor cells.     

A comparison of treatment of canine cyclic hematopoiesis with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF interleukin-3, and canine G-CSF.

Cyclic hematopoiesis in gray collie dogs is a stem cell disease in which abnormal regulation of cell production in the bone marrow causes cyclic fluctuations of blood cell counts. In vitro studies demonstrated that recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and granulocyte colony stimulating factor (G-CSF) all stimulated increases in colony formation by canine bone marrow progenitor cells. Based on these results, gray collie dogs were then treated with recombinant human (rh) GM-CSF, IL-3, or G-CSF subcutaneously to test the hypothesis that pharmacologic doses of one of these hematopoietic growth factors could alter cyclic production of cells. When recombinant canine G-CSF became available, it was tested over a range of doses. In vivo rhIL-3 had no effect on the recurrent neutropenia but was associated with eosinophilia, rhGM-CSF caused neutrophilia and eosinophilia but cycling of hematopoiesis persisted. However, rhG-CSF caused neutrophilia, prevented the recurrent neutropenia and, in the two animals not developing antibodies to rhG-CSF, obliterated periodic fluctuation of monocyte, eosinophil, reticulocyte, and platelet counts. Recombinant canine G-CSF increased the nadir neutrophil counts and amplitude of fluctuations at low doses (1 micrograms/kg/d) and eliminated all cycling of cell counts at high doses (5 and 10 micrograms/kg/d). These data suggest significant differences in the actions of these growth factors and imply a critical role for G-CSF in the homeostatic regulation of hematopoiesis.     

Defective polymorphonuclear leukocyte metabolism and function in canine cyclic neutropenia.

Humans and grey collie dogs with cyclic neutropenia are known to suffer from an increased rate of bacterial infection. Because of the previously described microanatomic abnormalities of lysosome formation found in the polymorphonuclear leukocytes (PMNs) of dogs with canine cyclic neutropenia, studies of these cells were undertaken. PMNs from grey collie dogs were found to have significant metabolic and functional abnormalities when compared with normal collie PMNs. These included abnormally increased postphagocytic C1-glucose oxidation, decreased iodination of trichloroacetic acid-precipitable protein in the resting and phagocytizing state, decreased levels of intracellular myeloperoxidase,and a bactericidal defect against a variety of bacteria. Phagocytosis was normal. These abnormalities appear to differ from those previously described in the PMNs of patients with chronic granulomatous disease of childhood and the Chediak-Higashi syndrome and more closely resemble those seen in hereditary myeloperoxidase deficiency. Thus, the studies reported here demonstrate defective PMN function in a disease state previously believed to be a model only of periodic hematopoiesis.     

Long-term treatment of canine cyclic hematopoiesis with recombinant canine stem cell factor

Grey collie dogs have cyclic fluctuations in their blood cell counts caused by a regulatory defect of hematopoietic stem cells. To examine the role of stem cell factor (SCF) or its receptor in this disorder, we investigated the stimulatory effects of recombinant canine SCF (rc-SCF) on in vitro marrow cultures, cloned and sequenced the grey collie SCF gene, and treated three grey collies with rc-SCF, either alone or in combination with recombinant canine granulocyte colony-stimulating factor (rcG-CSF). Colony-forming unit granulocyte-macrophage formation from grey collie or normal dog marrow showed similar dose-response curves for rc-SCF. Cloning and sequencing the SCF gene for two grey collies showed no evidence of mutations in the coding region of the SCF gene. Treatment with rc-SCF (10 to 100 micrograms/kg/d) did not induce neutrophilia except at the highest dose (100 micrograms/kg/d), but daily rc-SCF abrogated the neutropenic periods in doses of 20 micrograms/kg/d or greater. Combination of rc-G-CSF (0.5 to 1.0 microgram/kg/d) with rc-SCF treatment (20 to 50 micrograms/kg/d) suggested a synergistic effect, ie, the neutrophil levels on combined therapy were higher than the sum of the levels when these two cytokines were given separately. Long-term treatment of these dogs with rc-SCF in doses of 10 to 30 micrograms/kg/d was generally well tolerated, suggesting that SCF may be useful as a therapy for some chronic hypoproliferative disorders of hematopoiesis.     

Heterogeneity of bone marrow-directed immune mechanisms in the pathogenesis of neutropenia of felty's syndrome

Twenty-seven patients with Felty's syndrome were studied by the colony forming unit in culture (CFU-C) assay for possible immune mechanisms within the bone marrow compartment that could contribute to the neutropenia. Depletion of bone marow suppressor T cells resulted in normal CFU-C numbers in 7 patients. In 5 patients serum antiprecursor cell activity was detected. Blood monocytes failed to generate colony stimulating factor in 5 patients. Bone marrow-directed immune mechanisms are heterogeneous and could play a role in the pathogenesis of neutropenia in some patients with Felty's syndrome.     

Human cyclic neutropenia transferred by allogeneic bone marrow grafting

Human cyclic neutropenia shows many features in common with the animal model of cyclic neutropenia in grey collie dogs. Until now, however, evidence was lacking that cyclic neutropenia in man as in the dog is caused by a defect in a transplantable hematopoietic stem cell. A patient is presented who, while undergoing bone marrow transplantation as treatment for acute lymphoblastic leukemia in relapse, acquired cyclic neutropenia from her histocompatible sibling donor.     

Lithium is an ineffective therapy for human cyclic hematopoiesis

Cyclic hematopoiesis is a rare disease in man in which severe neutropenia recurs at 21-day intervals with associated illness. Because lithium carbonate therapy has been shown to eliminate cyclic hematopoiesis in grey collie dogs, we examined the effects of lithium treatment on five patients with this disease. With lithium levels maintained between 0.5 and 1.0 meq/liter, these patients showed no change in the fluctuations of their neutrophil counts. We conclude that lithium carbonate is not a simple cure for human cyclic hematopoiesis.     

Inhibition of bone marrow myeloid precursor cell proliferation by chemotactic oligopeptides

Three N-formylated oligopeptides with different known activities as chemotactic factors for leukocytes were studied to determined if these mediators affect the in vitro proliferation of myelomonocytic colony- forming cells (CFU-C) recovered from murine bone marrow. All three oligopeptides inhibited CFU-C growth in a dose-dependent fashion that correlated with their relative potencies as chemotactic factors. This inhibition was not altered by growth of CFU-C in the presence of indomethacin, by varying the concentrations of colony-stimulating factor (CSF), or by depleting marrow cell preparations of mature granulocytic elements. These studies indicate that chemotactic factors may mediate myelosuppression through effects on committed myeloid precursor cells in the marrow.     

Lithium augments GM-CSA generation in canine cyclic hematopoiesis

Cyclic hematopoiesis in gray collie dogs can be cured by lithium treatment. We examined the mechanism of lithium's effect by developing an assay for the canine equivalent of GM-CSF (called GM-CSA). Phytohemagglutinin (PHA)-stimulated canine blood mononuclear cells produce GM-CSA in a dose-dependent manner; this GM-CSA stimulates more neutrophil-containing colonies than does endotoxin-treated dog serum. Production of GM-CSA by PHA-stimulated normal dog cells was not altered by lithium. However, cells from gray collies during their neutrophilic period increased their GM-CSA when lithium (2 mEq/L) was added to low doses of PHA, whereas neutropenic gray collie cells did not. These data suggest that lithium could modulate cyclic hematopoiesis by increasing intramedullary GM-CSA at the time when marrow neutrophilic progenitor cells are at their nadir.     

In vitro interactions of PGE and cAMP with murine and human erythroid precursors

Addition of prostaglandins of the E series (PGE1, PGE2) in methylcellulose cultures of murine marrow results in a dose-dependent inhibition of the cloning efficiency of both BFU-E and CFU-C. However, CFU-E growth is unaffected. The inhibitory action of PGE is progressively overcome by increasing amounts of colony-stimulating factor (CSF), and with some limitations, also of erythropoietin (Ep). Addition of PGF2 alpha' associated or not with indomethacin, does not exert any significant effect on these hemopoietic precursors. In an attempt to unvail the mechanism(s) underlying these phenomena, dibutyryl-cyclic AMP (db-cAMP), theophylline (an inhibitor of phosphodiesterase), or theophylline + PGE were plated at various concentrations. Both db-cAMP and theophylline induce an inhibitory influence on both BFU-E and CFU-C growth, which mimicks that by PGEs; additionally, theophylline potentiates the inhibitory action of PGE1. In all these studies, the CFU-E number was not significantly modified. PGE action on BFU-E proliferation is clearly species-dependent, since PGE1 addition to human marrow methylcellulose cultures induces a significant enhancement of the number of both BFU-E and CFU-E derived colonies. This action was abolished upon removal of adherent cells, thus suggesting that PGE1 evokes a release of factor(s) enhancing human erythroid colony growth by adherent cells.     

Cyclic Hematopoiesis in Grey Collie Dogs: A Stem-Cell Problem

Studies were made to ascertain the possible basis of cyclic hematopoiesis in thegrey collie. The results of ³H-thymidineand ⁵⁹Fe labeling in conjunction with othermarrow parameters and peripheral countssuggest that the regular periodicity ofblood neutrophil and reticulocyte levelsis caused by a defect at the stem-cell—marrow interface. It is postulatedthat the ebb and flow of hemic cellproduction in the grey collie reflects competition for a limiting number of pluripotential stem cells with the alternatingcompetitive pressure provided by activation and deactivation of a neutrophil feedback circuit from periphery to stem cell.This mechanism can account for the characteristic 12-day periodicity and contributes to the phase difference in thecycling pattern of neutrophils relative toreticulocytes.

Submitted on January 26, 1973 Revised on June 15, 1973 Accepted on June 16, 1973     

Hairy cell leukemia: effect of 'hairy' cells on normal granulopoiesis in vitro.

The effect of 'hairy cells' (HC) on proliferation of normal marrow granulocytic progenitor cells (CFU-C) was studied in vitro, using cells obtained from the blood, spleen, and bone marrow from eight patients with hairy cell leukemia (HCL). Hairy cells of hairy-cell-conditioned media (HCCM) did not stimulate proliferation of normal CFU-C. Furthermore, HC and HCCM did not inhibit the growth of CFU-C induced by normal colony stimulating factor (CSF) or by feeder layers of normal blood leukocytes. These results confirm previous reports showing the inability of HC to produce CSF and suggest that inhibition of CFU-C proliferation, as measured by this technique, is not an important mechanism in the neutropenia observed in HCL.     

Correction of canine cyclic hematopoiesis with recombinant human granulocyte colony-stimulating factor

Canine cyclic hematopoiesis (CH) is an autosomal recessive disease of gray collie dogs that is characterized by neutropenic episodes at 14- day intervals. The biochemical basis for CH is not known but may involve a regulatory defect of the response to or production of a hematopoietic growth factor. Administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to two CH and one normal dog caused a marked leukocytosis (greater than 50,000 WBCs) in all three dogs. The leukocytosis was due largely to a greater than tenfold increase in neutrophils. Less pronounced but significant elevations in monocytes occurred during G-CSF treatment. The elevated WBC count was maintained for more than 20 days in all three dogs, and two predicted neutropenic episodes were prevented in both CH dogs during rhG-CSF treatment. A decline in the WBC count occurred simultaneously in all three dogs during the last five treatment days and was presumably associated with the development of neutralizing antibodies to the heterologous rhG-CSF protein. Bone marrow evaluation indicated that the swings in the myeloid/erythroid progenitor cells that are characteristic of CH were eliminated by rhG-CSF treatment in both CH dogs. These results suggest that the regulatory defect in canine CH can be temporarily alleviated by treatment with rhG-CSF and point to the potential treatment of human cyclic neutropenia with this agent.     

Studies on the regeneration of the CFU-C population in blood and bone marrow of lethally irradiated dogs after autologous transfusion of cryopreserved mononuclear blood cells

In a group of 8 lethally irradiated (1200 R) dogs, that were transfused autologously with cryopreserved mononuclear cells (MNC) derived from the peripheral blood by leucapheresis the concentration of colony-forming units in agar (CFU-C) in bone marrow and peripheral blood was estimated at regular intervals after irradiation and transfusion of MNC. The numbers of MNC transfused per kg body weight ranged from 0.32 x 10(9) to 1.63 x 10(9) with an incidence of CFU-C between 0.02 x 10(5) and 1.38 x 10(5). In 6 dogs the CFU-C levels in the bone marrow reached the normal pre-irradiation values between days 15 and 20. But in 2 dogs that had received the lowest CFU-C numbers the regeneration of the bone marrow CFU-C was markedly delayed. In general the time course of the bone marrow repopulation by CFU-C for single dogs was reflected by a corresponding regeneration pattern of the blood CFU-C. The time course of the curves for the blood CFU-C levels on the other hand was of the same kind as for the granulocyte values in the peripheral blood, thuations were seen in the blood CFU-C levels of single dogs before irradiation and after mononuclear leucocyte transfusion. Despite of such limitations the blood CFU-C content appeared to be a useful indicator of haematopoietic regeneration of the bone marrow.     

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.     

Amphotericin inhibition of hematopoiesis in vitro

The effect of amphotericin B on human and murine hematopoiesis was studied in vitro using assays for erythroid and myeloid colony-forming cells. Amphotericin consistently inhibited colony formation by erythroid and granulocyte-monocyte progenitor cells. Clear effects were observable at amphotericin concentrations of 1.0 μg/ml, and concentrations of 2.0 μg/ml caused approximately 50-60% inhibition of cloning of both murine and normal human bone marrow. These data suggest that amphotericin, in concentrations achieved in therapy, can impair hematopoiesis by a direct effect on precursor cells.     

Monocytes enhance gamma-interferon-induced inhibition of myeloid progenitor cell growth through secretion of tumor necrosis factor

In this study, we have examined the effects of autologous monocytes and T-lymphocytes on gamma-interferon (gamma-IFN)-induced inhibition of granulocyte-monocyte progenitor cells (CFU-GM) in vitro. Depletion of adherent cells from the mononuclear fraction of normal bone marrow (NBM) resulted in a significant reduction in the inhibitory effects of gamma-IFN on CFU-GM growth, whereas T-lymphocyte depletion had no effect. Adding back autologous monocytes to the underlayer fraction of agar culture resulted in a concentration-dependent enhancement of gamma-IFN-induced CFU-GM inhibition that did not require cell-cell contact. Adding back autologous T-lymphocytes had no effect and did not synergize with monocytes in enhancing gamma-IFN-induced inhibition. Based on the use of indomethacin and the pattern of CFU-GM subset growth, it was determined that prostaglandin E was unlikely to be the humoral inhibitory factor involved in this process. However, the effects of monocytes were completely reversed in the presence of a neutralizing monoclonal antibody to tumor necrosis factor (TNF), suggesting that monocyte-derived TNF was responsible for the enhancement of gamma-IFN-induced CFU-GM inhibition. This observation was further supported by the ability of gamma-IFN to induce an eightfold increase of baseline monocyte TNF secretion in agar culture. These data suggest that gamma-IFN may inhibit progenitor cell growth in vitro through indirect humoral mechanisms involving monocyte-derived TNF, as well as through direct inhibitory effects on CFU-GM proliferation. Because monocytes are a component of the bone marrow microenvironment, the ability of gamma-IFN to induce biologically relevant levels of monocyte-derived TNF may play an important role in the negative regulation of hematopoiesis.     

The effect of peripheral blood adherent cells from patients with aplastic anemia on in vitro hematopoiesis

To investigate the pathogenetic significance of peripheral blood adherent cells in aplastic anemia, the inhibitory effects of adherent cells obtained from the peripheral blood of 10 patients with aplastic anemia on the growth of CFUE and CFUC were compared with those of normal controls. The inhibitory effects of adherent cells were studied by means of co-culture with normal allogeneic bone marrow cells. The suppression of these inhibitory effects was also examined by adding indomethacin to the co-culture system. In addition, PGE2 concentrations in the adherent cell-conditioned media were assayed. The inhibition of the colony growth by peripheral blood adherent cells from the aplastic anemia patients was not significantly different from controls. Both the suppression of the inhibitory effects by indomethacin and the level of PGE2 production showed no significant difference between the patients and controls.