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.     

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.     

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.     

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.     

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.     

Cyclic neutropenia in mammals

Cyclic neutropenia (CN) has been well documented in humans and the gray collie. A recent model of the architecture and dynamics of hematopoiesis has been used to provide insights into the mechanism of cycling of this disorder. It provides a link between the cycling period and the cells where the mutated ELA2 is expressed. Assuming that the biologic defect in CN is the same in dogs, and the observation that the structure of hematopoiesis is invariant across mammals, we use allometric scaling techniques to correctly predict the period of cycling in the gray collie and extend it to other mammals from mice to elephants. This work provides additional support for the relevance of animal models to understand disease but cautions that disease dynamics in model animals are different and this has to be taken into consideration when planning experiments.     

Mechanism of canine cyclic hematopoiesis: the role of prostaglandin E in feedback regulation

Prostaglandin E inhibits granulocyte-macrophage colony formation in vitro in man and mouse, suggesting that it plays a role in feedback regulation of granulocyte production in vivo. Therefore, we examined the role of PGE in normal canine hematopoiesis and its potential role in the pathogenesis of cyclic hematopoiesis in grey collie dogs. The prostaglandin synthesis inhibitors indomethacin and ibuprofen (10(-5) M) increased CFU-C growth to 194 and 160% of control, respectively, while PGE2 addition caused a dose-dependent inhibition of bone marrow CFU-C growth in both normal and grey collie dogs. These concentrations of indomethacin and ibuprofen decreased bone marrow cell elaboration of PGE measured by radioimmunoassay to less than 5% of control values. The levels of PGE in leukocyte conditioned medium prepared from grey collies correlated with the number of monocytes in the conditioning cell suspension (r = 0.78, n = 10, p less than 0.05) so that PGE production per monocyte was no different in normal and grey collie dogs. The effect of PGE2 upon CFU-C was to inhibit formation of macrophage, but not neutrophil colony subtypes. These findings make aberrant PGE-mediated inhibition of precursor cells an unlikely mechanism to cause cyclic hematopoiesis, and show that PGE produced by monocytes acts as a feedback inhibitor for precursor cells destined to produce monocytes but not for those destined to form neutrophils.     

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.     

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.     

Treatment of canine cyclic neutropenia by lentivirus-mediated G-CSF delivery

Cyclic neutropenia is a rare disease that occurs both in humans and gray collie dogs and is characterized by recurrent severe neutropenia leading to bacterial infections and shortened life expectancy. Daily injections of recombinant granulocyte colony-stimulating factor (rG-CSF) are effective in shortening the period of severe neutropenia and reducing infections. After demonstrating that rG-CSF induced elevated neutrophil production in an affected dog, cytokine administration was stopped and 109 infectious units (IUs) of a lentivirus pseudotyped with vesicular stomatitis virus G protein (VSV-G) encoding canine G-CSF cDNA was administered intramuscularly. Serial blood cell counts showed elevated neutrophil production for longer than 17 months. Although neutrophil counts continued to cycle, the range at nadirs was from 3710 to 5300 cells/microL, well above the nadirs before lentivirus administration. After the injection of lentivirus, mean neutrophil counts +/- SD were 12 460 +/- 4240 cells/microL, significantly increased over both pretreatment values of 3040 +/- 2540 cells/microL(P <.0001) and neutrophil counts during G-CSF administration of 10 290 +/- 4860 cells/microL(P <.007). The changes in blood counts from lentivirus injection were associated with absence of clinical signs of infection and fever. The gray collie continued to gain weight and was no longer housed in a pathogen-free environment. Genomic DNA from muscle at injection sites was positive for provirus, whereas gonad, lung, spleen, heart, liver, kidney, leukocytes, and noninjected muscle samples were all negative for provirus. Thus, intramuscular administration of lentivirus encoding G-CSF provided sustained therapeutic levels of neutrophils, suggesting this approach may be applied for long-term treatment of patients with cyclic and other neutropenias.     

Lithium stimulation of granulopoiesis in diffusion chambers--a model of a humoral, indirect stimulation of stem cell proliferation

Lithium has been recognized as a stimulator of granulopoiesis both in vivo and in vitro. The mechanism by which lithium provokes this stimulation is unclear, with previous data focusing on such divergent causes as direct effects on progenitor cells v elevations in granulocyte macrophage colony-stimulating activity (GM-CSA) production. In the present study, we used a model system of granulopoiesis in diffusion chambers to study this stimulation of granulopoiesis. Lithium pretreatment of mice followed by a rest period to allow for excretion of the lithium (confirmed by serum assays) revealed a stimulation of progenitor cell growth within the diffusion chambers. No changes in the serum and chamber fluid GM-CSA levels were discernible between the control host mice and the lithium-pretreated mice. These data indicate that lithium stimulates granulopoiesis by an indirect mechanism that does not appear to involve GM-CSA.     

Factors influencing release of granulocyte-macrophage colony-stimulating activity from human mononuclear phagocytes

Mononuclear phagocytes play an important role in the regulation of hematopoiesis, not only by producing regulatory monokines such as prostaglandins, tumor necrosis factor and interleukin-1 (IL-1), but also by the production of colony-stimulating activity (CSA). Previously, we have demonstrated that granulocyte-macrophage CSA (GM-CSA) production by mononuclear phagocytes can be induced by IL-1. In the present study, the influence of culture conditions on the production of GM-CSA was studied. It was found that both human sera and fetal bovine sera contain constituents - at present undefined - that induce GM-CSA production. These factors are distinct from IL-1 and lipopolysaccharide. In selected experiments, no GM-CSA-inducing effect of serum was found, suggesting that the effect may be donor-related. GM-CSA release in the presence of serum could be reduced by 40% after incubation of mononuclear phagocytes at low cell concentrations in methylcellulose, indicating that intimate cell-cell contact is an additional factor that enhances GM-CSA release.     

Production of erythroid burst-promoting activity (BPA) and granulocyte-monocyte colony-stimulating activity (GM-CSA) by isolated human T-lymphocyte subpopulations

T-lymphocytes and monocytes are prominent among the classes of normal human cells that have been implicated in the production of the hemopoietic growth factors granulocyte-macrophage colony-stimulating activity (GM-CSA) and erythroid burst-promoting activity (BPA). To investigate the nature of the cooperativity that occurs during the elaboration of these growth factors by activated T-lymphocytes and monocytes in vitro, and to define the subsets of T cells involved in this response, we studied the production of GM-CSA and BPA by populations of T-lymphocytes isolated by fluorescence-activated cell sorting, using the monoclonal antibodies OKT3, OKT4, and OKT8. When OKT3+, OKT4+, or OKT8+ cells were incubated for five days in liquid suspension cultures, their production of GM-CSA and BPA was undetectably low. When 5% autologous monocytes were added to the cultures, no increase in the secretion of either of these classes of growth factors was noted. In the presence of concanavalin A (Con A), measurable quantities of both GM-CSA and BPA were elaborated by all three populations of T cells in the absence of monocytes; however, when autologous monocytes were added to the Con A-stimulated T cells, the secretion of both GM-CSA and BPA was markedly enhanced. In addition, we found that supernates of unfractionated T cells incubated with Con A contained not only GM-CSA and BPA but also a potent inhibitor(s) of BPA that could be demonstrated by dilution of the media and removed by gel filtration. In contrast, no inhibitor of GM-CSA was found. By molecular sieve chromatography of the supernates, GM-CSA and BPA coeluted as a single peak. However, the two biologic activities could be separated on the basis of heat stability, since GM-CSA proved to be heat labile whereas BPA did not. Our data indicate that GM-CSA and BPA derived from human T cells are similar in their apparent molecular weights and in the pattern of their production in suspension cultures in response to lectin stimulation. The secretion of both GM-CSA and BPA by Con A-stimulated T cells is facilitated by the presence of autologous monocytes, and is not restricted to either the OKT4- or the OKT8-defined subset.     

Effects of recombinant granulocyte colony-stimulating factor treatment on hematopoietic cycles and cellular defects associated with canine cyclic hematopoiesis.

Canine cyclic hematopoiesis (CH) is an autosomal recessive disease of gray collie dogs that is characterized by 14-day cycles of neutropenia, monocytosis, thrombocytosis, and reticulocytosis. Platelets from CH dogs have decreased dense-granule serotonin pools and decreased aggregation responses to collagen, platelet-activating factor (PAF), and thrombin. Recombinant granulocyte colony-stimulating factor (rG-CSF) was administered (5 micrograms/kg, b.i.d.) to four CH and six normal dogs to determine if G-CSF therapy corrected qualitative platelet defects in CH dogs. Neutrophil counts increase to greater than 25,000 cells/microliters within 24 h after starting treatment in all dogs. Treatment with G-CSF blocked neutropenic episodes in the CH dogs. Platelet aggregation, and serotonin content and secretion were significantly (p less than 0.05) decreased in the CH dogs both before and during recombinant human (rh) G-CSF treatment compared to normal dogs. Neutrophil myeloperoxidase, a primary granule enzyme, was significantly (p less than 0.05) decreased in CH dogs and was not corrected by rhG-CSF treatment. Administration of rG-CSF to CH dogs eliminated cell cycles but apparently did not correct cellular defects in CH dogs. Identification of primary biochemical defects in cells from CH dogs may be crucial to investigating the biochemical basis for cyclic hematopoiesis.     

Breed distribution and history of canine mdr1-1Delta, a pharmacogenetic mutation that marks the emergence of breeds from the collie lineage

A mutation in the canine multidrug resistance gene, MDR1, has previously been associated with drug sensitivities in two breeds from the collie lineage. We exploited breed phylogeny and reports of drug sensitivity to survey other purebred populations that might be genetically at risk. We found that the same allele, mdr1-1Delta, segregated in seven additional breeds, including two sighthounds that were not expected to share collie ancestry. A mutant haplotype that was conserved among affected breeds indicated that the allele was identical by descent. Based on breed histories and the extent of linkage disequilibrium, we conclude that all dogs carrying mdr1-1Delta are descendants of a dog that lived in Great Britain before the genetic isolation of breeds by registry (ca. 1873). The breed distribution and frequency of mdr1-1Delta have applications in veterinary medicine and selective breeding, whereas the allele's history recounts the emergence of formally recognized breeds from an admixed population of working sheepdogs.     

Decreased hematopoietic accessory cell function following bone marrow transplantation

Hematologic engraftment following bone marrow transplantation requires not only pluripotent stem cells but also functioning accessory cells whose trophic factors support the proliferation and differentiation of stem cells and progenitors to mature blood cells. To better understand the regulation of hematopoiesis following transplantation, we studied hemopoietic accessory cell function in bone marrow transplant recipients 3 weeks to 10 months following transplantation. In general, hematopoietic accessory cell function was decreased following bone marrow transplantation. Sequential fractionation of accessory cells demonstrated that adherent cells often produced near-normal functional burst-promoting activity (BPA) and granulocyte-macrophage colony-stimulating activity (GM-CSA), but Fc receptor-positive (Fc+) cells and T cells uniformly produced greatly reduced BPA and GM-CSA, as compared to transplant donor cells. This cellular deficiency was corrected by soluble burst-promoting activity and granulocyte-macrophage colony-stimulating activity and so appeared to be due to the failure of accessory cells to produce trophic hormones. Limiting-dilution analysis (LDA) for proliferating T-cell precursors demonstrated a greatly reduced frequency in phytohemagglutinin-responsive cells, supporting the role of deficient hematopoietic growth factor production by activated T cells in transplant recipients. This hemopoietic accessory cell defect may thus reflect more generalized lymphocyte dysfunction in these patients. Hematopoiesis following bone marrow transplantation appears to rely upon growth factors released by accessory cells in the adherent layer.     

Effect of plasmacytoma cells on the production of granulocyte-macrophage colony-stimulating activity (GM-CSA) in the spleen of tumor-bearing mice

Mice bearing syngeneic plasma cell tumors are characterized by elevated numbers of granulocyte-macrophage progenitors (GM-CFU) in the spleen. We investigated the role of syngeneic plasmacytomas in the hematologic response to tumor cell transplantation by assaying the production of granulocyte-macrophage colony-stimulating activity (GM-CSA) by cultured spleen cells of tumor-bearing mice and by plasmacytoma cells, alone and in coculture with spleen cells. Elevated levels of GM-CSA were detected in 7-day culture supernatants of spleen cells from Balb/c mice transplanted 2 weeks previously with syngeneic 4T00.1 plasmacytoma cells. Colony assays of spleen cells from tumor-bearing mice demonstrated the presence of both granulocyte-macrophage and tumor cell colonies. A high frequency of GM-CFU was detected in cultures which had not been supplemented with an exogenous source of GM-CSA. Significant levels of GM-CSA were detected in media conditioned by 4T00.1 plasmacytoma cells. 4T00.1-conditioned medium did not stimulate the growth of primative erythroid (BFU-E) and multilineage (CFU-GEMM) colonies, but stimulated the growth of FDC-P1 cells, thereby establishing the activity produced by 4T00.1 cells as GM-CSF. The levels of GM-CSA in media conditioned by coculturing control spleen and 4T00.1 cells were significantly higher than those detected in media conditioned by spleen cells alone. The colony frequency induced by the coculture supernatants, however, did not exceed the sum of the colonies detected in marrow cell cultures stimulated with media conditioned by control spleen and 4T00.1 cells alone. Our findings demonstrate that murine plasmacytoma cells are capable of secreting GM-CSF. They further suggest a key role for GM-CSA production by tumor cells in the hemopoietic response of mice bearing syngeneic plasma cell tumors.     

Signal requirements for the generation of T-lymphocytes from T-depleted bone marrow cells: a sequential study

We have investigated the sequence of signals provided by a B- and null-cell-derived prothymocyte-differentiating activity (PTDA), phytohemagglutinin (PHA), and interleukin 2 (IL2) to the generation of mature T-lymphocytes by T-depleted bone marrow (BM) cells. Sequential studies show that preincubation of CD2-, CD3-, CD4-, CD6-, and CD8- BM cells with PTDA, but not with recombinant (r) IL2 or PHA increased their capacity to proliferate in liquid culture and to form agar T-cell colonies provided both PHA and rIL2 were added to the cultures. In contrast, the growth of T-cell-containing BM was significantly enhanced in both liquid and agar culture following its preincubation with rIL2 as well as with PTDA. The selective effect of PTDA on CD2-, CD3-, CD4-, CD6-, CD8- BM cells was abolished by adding a CD7 monoclonal antibody to the T-cell-purging coctail. Cell marker studies performed on T-cell-depleted BM-derived liquid or agar cultures have shown that they contain up to 70%-85% CD2+, CD3+, CD4+, CD8- cells. No IL1 or IL2 could substitute for PTDA, nor have these activities, as well as interferon (IFN), IL3, IL4, or granulocyte-macrophage colony-stimulating activity (GM-CSA) been detected so far in PTDA-containing preparations. These results indicate that PTDA can trigger marrow T-cell precursors into PHA-responsive T cells, which, following activation by PHA, require IL2 for growth. It is suggested that this may represent a thymus-independent alternative pathway for T-cell differentiation and activation.     

The production of colony stimulating activity by monocyte enriched fractions from murine continuous bone marrow culture adherent layers

The production of colony stimulating activity (GM-CSA) within murine continuous bone marrow cultures was investigated by subjecting either the nonadherent or the adherent layer cells to separation by velocity sedimentation. The presence of GM-CSA in conditioned medium was defined by the support of granulocyte/macrophage colony formation in soft agar culture. Cell free conditioned medium from weekly feedings of intact continuous marrow cultures and medium conditioned by each fraction of velocity sedimentation separated, nonadherent cells did not contain assayable GM-CSA. However, medium conditioned by fractions of adherent layer cells with a modal sedimentation velocity of 8.8 mm/h (range 6.2-10.6 mm/h) contained GM-CSA. Cytochemical studies with Wright's-Giemsa and non-specific esterase stains in addition to immunofluorescent studies with anti-collagen III, anti-collagen IV and monoclonal anti-Mac I antibodies to define fibroblasts, endothelial cells and monocytes, respectively, demonstrated that the cells within the GM-CSA producing fractions were enriched with monocytes/macrophages. Fibroblasts and a small proportion of endothelial cells were also present. GM-CSA is produced within the microenvironment (adherent layer) of murine continuous marrow cultures. Either adherent layer monocytes and/or a monocyte-endothelial cell interaction account for the GM-CSA production.     

Abnormal GM-CSA generation by lymphocytes in patients with cyclic hematopoiesis

Human cyclic hematopoiesis (CH) is a disease characterized by regular 21-day cyclic fluctuations of blood cell counts due to fluctuations in bone marrow cell production. The regular periodicity of the fluctuations suggests a defect in a regulatory feedback control loop. We examined the production of monocyte-derived recruiting activity (MRA) by monocytes and the response to MRA of lymphocytes from three patients with CH. MRA production was normal or increased in patients' monocytes, but granulocyte-macrophage colony-stimulating activity (GM-CSA) production in response to MRA was decreased in lymphocytes from patients with CH (p = 0.005). These data suggest that the regulatory defect in CH may involve defective lymphocyte generation of GM-CSA, resulting in deficient production of mature neutrophils.