5-fluorouracil-induced thrombocytosis in mice is independent of the spleen and can be partially reproduced by repeated doses of cytosine arabinoside.

Experiments were done to characterize the pronounced and prolonged thrombocytosis that develops in mice during recovery from the marrow hypoplastic effects of a single injection of 5-fluorouracil (5-FU). Measurements were made of platelet and megakaryocyte numbers, size of mature megakaryocytes, marrow cellularity, hematocrit, and reticulocytes. Mice that had been splenectomized a month before receiving 5-FU displayed the same pattern of thrombocytosis as intact mice, so a role for the spleen in its genesis or persistence could not be identified. During recovery from two or three doses of cytosine arabinoside (Ara-C), given at intervals of 2 days, thrombocytosis and megakaryocytosis of similar magnitudes to those seen after 5-FU occurred. Bone marrow cellularity, hematocrits, and reticulocytes were identical after three doses of Ara-C or one dose of 5-FU. Megakaryocytes were initially macrocytic during recovery from the hypoplastic thrombocytopenia produced by 5-FU or Ara-C. These similarities to effects of repeated doses of Ara-C suggested that some of the effects of 5-FU were due to its prolonged action in vivo, which could cause sequential killing of proliferating cells. Abnormalities of megakaryocytes and platelets reversed promptly when normal or increased numbers of megakaryocytes and/or platelets were produced after Ara-C, but they persisted in mice that received 5-FU. This difference suggested that regulation of megakaryocyte progenitors was perturbed during recovery from 5-FU.

Megakaryocytes increase in size within ploidy groups in response to the stimulus of thrombocytopenia.

Experiments were done to determine if sizes of megakaryocytes within a defined maturation stage were strictly determined by amount of nuclear DNA. Normal mice, mice recovering from an acute episode of thrombocytopenia induced by a single injection of heterologous antiplatelet serum (APS), and mice with sustained thrombocytopenia from daily injections of APS were examined. Areas of mature megakaryocytes were measured in bone marrow smears stained with polychromatic stains. The nuclear DNA content of the same cells was then measured microspectrophotometrically after staining by the Feulgen reaction. Normal megakaryocytes showed a trimodal, lognormal distribution of nuclear chromophore, corresponding to 8n, 16n, and 32n with smaller numbers of 4n and 64n cells; 16n was the predominant ploidy class. In response to thrombocytopenia, ploidy values shifted: the proportions of 8n and 16n cells decreased; 32n and 64n cells increased; 128n megakaryocytes occasionally appeared. These shifts were accompanied by an increase in the average size of all megakaryocytes. In addition to shifts to higher ploidy values, megakaryocytes within ploidy groups became larger than normal megakaryocytes of the same ploidy especially in the mice with sustained thrombocytopenia. These findings show that megakaryocyte size in thrombocytopenic mice is influenced by factors other than the ploidy and maturity of the cell.

Thrombocytopoietic response to immunothrombocytopenia in nude mice.

Thrombocytopoiesis was evaluated in T cell-deficient nu/nu mice and in T cell-replete nu/+ controls to determine if abnormalities would be associated with the deficiency of T cells. Mice were studied in the unperturbed steady state and after acute immunothrombocytopenia was induced by an injection of guinea pig antimouse platelet serum (APS). The state of thrombocytopoiesis was determined from platelet counts, megakaryocyte size, megakaryocyte number, and numbers of Meg-CFC. Splenic lymphocytes were evaluated by response to the mitogens bacterial lipopolysaccharide (LPS), phytohemagglutinin (PHA), and concanavalin A (Con A). Hematocrits, reticulocyte counts, leukocyte counts, marrow cellularity, GM-CFC, and BFU-E also were measured. Steady state thrombocytopoiesis was identical in nu/nu and nu/+ mice. In response to an injection of APS, acute thrombocytopenia was followed by macromegakaryocytosis and rebound thrombocytosis in mice of both genotypes. Splenic Meg-CFC increased in nude mice after APS or an injection of normal guinea pig serum (NGpS), and splenic GM-CFC increased after APS. Neither Meg-CFC nor GM-CFC increased in the spleens of nu/+ mice, but they showed early transient increases in bone marrow that did not occur in nu/nu mice. Sporadic, but weak, mitogenic responses to PHA or Con A were occasionally observed with nu/nu spleen cells, but these did not correlate with the state of thrombocytopoiesis. The results demonstrated that platelet production was normal in nu/nu mice and that megakaryocytopoiesis and platelet production responded to the stimulus imposed by acute immunothrombocytopenia. Increases in megakaryocyte size and platelet production occurred independently of changes in numbers of Meg-CFC, GM-CFC, or BFU-E. A normal complement of T cells appears to be unnecessary for normal platelet production and its augmentation in response to the stimulus of acute immunothrombocytopenia in vivo.

Postirradiation thrombocytopoiesis: suppression, recovery, compensatory states, and macromegakaryocytosis.

Two unusual features of the regulation of megakaryocytopoiesis have been found in irradiated mice. The first is that the response to thrombocytopenia loses its specificity for thrombocytopoiesis when the thrombocytopenia is induced at the time of exposure to sublethal doses of radiation. Under these conditions, there is stimulation of both thrombocytopoiesis and erythropoiesis. The second unusual feature is that a completely or partially compensated hypomegakaryocytic state may develop after "recovery" from the earlier severe postirradiation myelodepression. Occurrence of this condition is not dependent on the presence or absence of the spleen. It is characterized by a dissociation between platelet and megakaryocyte numbers, with platelets being relatively higher. There is an associated increase in mean megakaryocyte size. Both the megakaryocytopenia and macromegakaryocytosis are due to a deficiency of smaller megakaryocytes in the marrow. The postirradiation abnormality of megakaryocyte size distribution can not be accounted for by irradiation-induced abnormalities of either hemopoietic or stromal cells. The degree of megakaryocytic macrocytosis does not correlate with the platelet or megakaryocyte count after recovery from sublethal irradiation or after recovery from lethal irradiation and rescue with normal bone marrow cells. Megakaryocytic macrocytosis, as identified by an increase in average size of mature cells, occurs in response to thrombocytopenia and in several hypomegakaryocytic states in which thrombocytopenia is absent or is mild in degree. Comparison of size distribution curves, analysis of sizes of immature megakaryocytes, and determination of the stability of the megakaryocyte count indicate that different mechanisms probably prevail. The presence or absence of thrombopoietin or other megakaryocyte growth factors in these conditions may provide clues about the mechanisms.

Functionally abnormal stromal cells and megakaryocyte size, ploidy, and ultrastructure in Sl/Sld mice.

The first goal of the present studies was to determine if Sl/Sld megakaryocytes have features in common with the macrocytic megakaryocytes that genetically normal mice produce in response to acute platelet depletion. The second was to test the hypothesis that megakaryocyte abnormalities in Sl/Sld mice are due to genetically determined hemopoietic stromal cell abnormalities. Sizes and ploidies of mature Sl/Sld megakaryocytes were measured. Macrocytosis and a shift to higher ploidy values were found compared with normal. Within ploidy groups 16N-64N, Sl/Sld megakaryocytes were larger than normal megakaryocytes of the same ploidy. Transmission electron microscopy revealed that Sl/Sld megakaryocyte nuclei contain more and larger nucleoli, and the chromatin was more dispersed than in normal megakaryocyte nuclei of comparable maturity. Asynchronous megakaryocyte cytoplasmic maturation was found. Sl/Sld macrophages were also ultrastructurally abnormal. Megakaryocytic macrocytosis was reproduced in long-term bone marrow cultures in which the adherent layer was formed by Sl/Sld cells. It was the same if cultures were recharged with Sl/Sld or +/+ hemopoietic cells. Previously reported ambiguities in mixed cell cultures were avoided by recharging the adherent layers with only a million cells. These results were correlated with previously published observations. Sl/Sld megakaryocytes have features in common with megakaryocytes from acutely thrombocytopenic animals. One feature, macrocytosis, appears to be due to abnormal Sl/Sld stromal cells that are reproduced as adherent layer cells in long-term cultures. The responsible stromal cells in Sl/Sld mice may be counterparts of megakaryocytopoietic regulatory cells in the marrow stroma of normal animals.

Independence of megakaryocyte number and size in long-term cultures of normal mouse marrow.

Megakaryocytopoiesis was evaluated in long-term cultures of normal murine marrow to determine whether the number and size of megakaryocytes were independent or interdependent. Numbers of megakaryocytes and granulocytes varied widely in different experiments, due, in part, to varying concentrations of hydrocortisone in the culture medium. The sizes of acetylcholinesterase-positive cells were the same in cultures with as much as a 20-fold difference in megakaryocyte numbers. These results indicate that, in the closed culture system containing normal stromal cells, megakaryocyte size and number are not reciprocal as they were in many previously reported cultures of S1/S1d mouse marrow. The results suggest that separate stromal functions may determine precursor cell proliferation and nuclear endomitosis in megakaryocytes in vitro. The relationship of these findings to regulation of megakaryocytopoiesis in vivo remains speculative.

Modulation of radiation-induced hemopoietic suppression by acute thrombocytopenia.

Modifications of radiation-induced hemopoietic suppression by acute thrombocytopenia were evaluated. Immediately before or after exposure to sublethal irradiation, mice were given a single injection of anti-mouse platelet serum (APS), normal heterologous serum, neuraminidase (N'ase), or saline, or no further treatment was provided. Hemopoiesis was evaluated by blood cell counts, hematocrits, and incorporation of [75Se]selenomethionine into platelets. APS and N'ase induced an acute thrombocytopenia from which there was partial recovery before the platelet count started to fall from the radiation. During the second post-treatment week, both thrombocytopoiesis and erythropoiesis were greater in mice that received APS or N'ase in addition to radiation than in control irradiated mice. Differences in leukopoiesis were not apparent. Therefore, both thrombocytopoiesis and erythropoiesis appeared to be responsive to a stimulus generated by acute thrombocytopenia in sublethally irradiated mice.

Macrocytic megakaryocytes in cultures of S1/S1d bone marrow.

The thrombocytopoietic system of S1/S1d mice is characterized by macromegakaryocytosis and megakaryocytopenia, but the mechanisms responsible for reciprocal abnormalities of megakaryocyte number and size are unknown. These mice have a genetically determined abnormality of their hemopoietic microenvironment that can, in part, be reproduced as abnormal adherent stromal cells in bone marrow cultures. Cultures of bone marrow were therefore done to determine if the megakaryocytic abnormalities of S1/S1d marrow would also be reproduced in them. Cultures composed entirely of S1/S1d cells showed persistent macrocytosis of megakaryocytes when compared with cultures of normal +/+ marrow. At various times of culture, there were also reduced numbers of megakaryocytes, total cells, and granulocytes in the supernatants of S1/S1d cultures. Mixed cultures of S1/S1d and +/+ cells yielded inconclusive findings. The fact that macromegakaryocytosis occurred in cultures of S1/S1d marrow strongly suggests that its in vivo determinants were, in part, reproduced in culture.

Megakaryocytopoiesis in irradiated, splenectomized mice.

The hypomegakaryocytic state that develops after exposure to sublethal doses of ionizing radiation was evaluated in splenectomized and intact mice. The percentage reduction of marrow megakaryocytes was greater than that of platelets at comparable times post-irradiation. After initial recovery a secondary drop in platelet counts occurred earlier in intact than in splenectomized mice. The average size of mature megakaryocytes was found to be increased, due primarily to marked reductions in megakaryocytes of smaller size. These results indicate that the spleen acts more to reduce than to increase the platelet count after exposure to sublethal doses of whole body radiation and that megakaryocyte size may be increased by reduction in numbers of small megakaryocytes without an increase in large megakaryocytes.

Does autoregulation of megakaryocytopoiesis occur?

Although a major regulator of thrombocytopoiesis is the number of circulating platelets, several observations suggest that independent alternative regulatory mechanisms may exist. In some situations there is a curious association of megakaryocytopenia and megakarocytic macrocytosis in spite of normal platelet counts. If macrocytosis is considered as a sign of stimulation, this association suggests a cause and effect relationship between decreased numbers and increased size of megakaryocytes. This thesis was tested by examining the delayed effects of sublethal irradiation and the acute effects of hydroxyurea in mice. It was found that megakaryocytopenia and macromegakaryocytosis occurred together and that platelets counts were either normal or only slightly reduced. Therefore it was concluded that normal numbers of platelets could be produced by decreased numbers of megakaryocytes. Megakaryocytopenia appeared to be compensated, in part, by increased size of megakaryocytes, but the mechanism by which this occurred has not been elucidated. It is postulated that a reduction in the number of cells of the megakaryocytic system is sensed by a homeostatic mechanism that then acts to stimulate the cells that are present. This stimulation may then be manifested as macrocytosis of megakaryocytes.

Regulation of megakaryocytes in W/Wv mice.

W/Wv mice were injected with antiplatelet serum to produce thrombocytopenia or with platelet transfusions to induce thrombocytosis. The responses of their platelets and megakaryocytes were followed to determine if proliferative abnormalities of the megakaryocytic system would be detected. W/Wv mice responded normally to the stimulation from thrombocytopenia with rebound thrombocytosis, macromegakaryocytosis, and macrothrombocytosis. The megakaryocytes of these mice became smaller than normal in response to post-thrombocytopenic rebound thrombocytosis but not to transfusion-induced thrombocytosis. Thus, endogenous thrombocytosis appeared to be a more potent suppressor of megakaryocyte growth than exogenous. These results failed to reveal an effective abnormality of the thrombocytopoietic regulatory system of W/Wv mice in spite of their intrinsically reduced numbers of megakaryocytes and the well known defect of stem cell proliferation. Thrombocytopoietic regulation appeared, therefore, to occur mainly at the committed, rather then pluripotential, stem cell level, and normal responses of the platelet system were observed in spite of severe abnormalities at the pluripotential stem cell level.

Megakaryocytic responses to thrombocytopenia and thrombocytosis in Sl/Sld mice.

Sl/Sld mice maintain normal platelet counts in spite of a reduced number of megakaryocytes, but their megakaryocytes are macrocytic. The present studied were done to analyze platelet turnover in the steady state and thrombocytopoietic responses to perturbations of the platelet count. Platelet production, estimated with 35S incorporation in the steady state, blood volume, and total complement of peripheral platelets were normal. Sl/Sld mice responded to thrombocytopenia with normal degrees of macromegakaryocytosis and production of macrocytic platelets, but rebound thrombocytosis did not occur. Their megakaryocytes were unusually responsive to transfusion-induced thrombocytosis, showing a prompt and substantial reduction in size. Platelet turnover was normal in undisturbed Sl/Sld mice, and feedback regulation occurred. However, increased numbers of platelets were not produced in response to stimulation. The findings are consistent with the possibility that their megakaryocytes are large because of stimulation rather than a direct local effect of the abnormal microenvironment.

Thrombocytotic suppression of megakaryocyte production from stem cells.

Megakaryocytopoiesis in the spleens of lethally irradiated mice transplanted with marrow cells was suppressed by platelet transfusions. In one group of experiments, animals were irradiated and transfused with bone marrow cells on day O. They were then given either no treatment, platelets, platelet-poor plasma, or saline on days 0, 2, 4, 6, and 8, and then were sacrificed on day 10. Megakaryocytes per section in the spleens of mice receiving platelets were 24%-48% of the values in the groups given plasma, saline, or bone marrow only. The number of pure megakaryocyte colonies was also diminished by platelet hypertransfusion. Another experiment examined the effect of platelets or plasma administered on days 1 and 2 or days 6 and 7 after irradiation and bone marrow transfusion. Hypertransfusion on days 6 and 7 was as effective in suppressing megakaryocytopoiesis as hypertransfusion every other day for 10 days. Animals given platelets or plasma only on days 1 and 2 did not have any significant change in their megakaryocyte number. These results implied that committed megakaryocyte precursors were more sensitive to inhibition by increased platelet levels than pluripotential stem cells. Further experiments with plethoric animals indicated that different levels of erythropoietin did not account for the effects of platelet hypertransfusion. The findings could be explained by inhibition of cell proliferation or of differentiation of megakaryocyte precursors by increased platelet levels.

Effects of vincristine on normal and stimulated megakaryocytopoiesis in the rat.

Vincristine was given to rats in which thrombocytopoiesis was either normal or acutely or chronically stimulated by injections of heterologous antiplatelet serum. A single dose of 0.3 mg/kg was given intravenously. The drug produced an early and a delayed megakaryocytopenia suggesting that it was toxic to differentiated megakaryocytes as well as to proliferating stem cells. The results support the hypothesis that vincristine-induced thrombocytosis may be due to homeostatic adjustments which, in turn, are activated as a result of drug-induced cytotoxicity.