Failure of W/Wv mouse-derived cultured mast cells to enter S phase upon contact with NIH/3T3 fibroblasts

Although W/Wv mutant mice are profoundly deficient in tissue mast cells, these mice do have cells with similar features of mast cells that develop from their bone marrow cells as efficiently as those from congenic +/+ mice in pokeweed mitogen-stimulated spleen cell- conditioned medium (PWM-SCM). With cultured mast cells (CMCs), we analyzed the mechanism of mast-cell deficiency in tissues of W/Wv mice. CMCs were established from bone marrow cells of W/Wv and congenic +/+ mice with PWM-SCM, and then co-cultured with various mouse fibroblast cell lines without PWM-SCM. All the examined mouse embryo-derived fibroblast cell lines maintained CMCs derived from +/+ mice, but not CMCs from W/Wv mice, for greater than 2 weeks. Mast cells in S phase were observed only in CMCs derived from +/+ mice under these conditions. The poor survival of W/Wv CMCs as compared with +/+ CMCs was not owing to a differential death rate but to the inability of W/Wv CMCs to continue active proliferation on fibroblasts without PWM-SCM. By synchronizing CMCs at the G1 phase of the cell cycle, the defect in W/Wv CMCs was further characterized as a failure to transit G1 and enter the S phase upon contact with fibroblasts. This finding indicates the indispensable function of the W gene product(s) for this response.     

Megakaryocytopenia in W/Wv mice is accompanied by an increase in size within ploidy groups and acceleration of maturation

Megakaryocytopoiesis was evaluated in W/Wv mice and their normal +/+ littermates to analyze the mechanisms by which normal platelet production is maintained in W/Wv mice even though numbers of megakaryocytes are low. Relative sizes of megakaryocytes, and their nuclei and cytoplasm, were measured microscopically in bone marrow smears, and the ploidy of the same cells was measured by two-wavelength microspectrophotometry. Maturation rate of megakaryocytes was estimated after they were labeled with tritiated thymidine. W/Wv megakaryocytes were macrocytic: average cell size was increased in each ploidy group. The increase in cytoplasmic area exceeded that of the nucleus. Further analysis of the predominant 16N ploidy group revealed that the increase in average cell size was due to depletion of cells of small size. Megakaryocytes matured more rapidly than normal in W/Wv mice. These results showed that megakaryocyte size and ploidy can be regulated separately. They suggest that alterations in cell growth and maturation may be mechanisms by which the organism can compensate for a deficiency in numbers of megakaryocytes, but they do not define the mechanism by which the deficiency may be sensed or by which the compensatory changes may be mediated. This is a US government work. There are no restrictions on its use.     

Defective protective capacity of W/Wv mice against Strongyloides vatti infection and its reconstitution with bone marrow cells

The susceptibility of congenitally anemic, and mast cell deficient W/Wv mice to infection with Strongyloides ratti was examined. After a primary infection, W/Wv mice showed greater and more persistent peak larval counts than did normal littermates. Worm expulsion was also slower in W/Wv mice than in +/+ mice. Furthermore, difference in susceptibility was expressed as early as 24 h after infection, suggesting not only that protective mechanisms of the gut but also of the connective tissue were defective in W/Wv mice. Reconstitution with bone marrow or spleen cells from +/+ mice was effective in restoring the protective response in W/Wv mice, whereas thymocytes or mesenteric lymph nodes had no effect. Both connective tissue and mucosal mast cells were repaired in W/Wv mice after marrow reconstitution and infection. Since relatively long incubation period was required for the expression of such reconstituting activities, bone marrow cells seem to contain precursor cells of the effector and/or regulator cells.     

Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation.

Production of tissue mast cells was evaluated in genetically anemic mice of W/Wv genotype and was found to be abnormal. In the skin of adult W/Wv mice the number of mast cells/cm was less than 1% of the number observed in the congeneic +/+ mice. No mast cells were detectable in other tissues of the W/Wv mice. After transplantation of bone marrow cells from +/+ mice the number of mast cells in the skin, stomach, caecum, and mesentery of the W/Wv mice increased to levels similar to those of the +/+ mice. These results show that the W/Wv mouse is a useful tool for the investigations concerning the physiologic roles and the origin of mast cells.     

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.     

Erythrocyte replacement precedes leukocyte replacement during repopulation of W/Wv mice with limiting dilutions of +/+ donor marrow cells.

The severe macrocytic anemia of the stem-cell-deficient W/Wv mouse is alleviated by intravenous injection of normal marrow cells. Donor cells replace the host erythrocytes, but the fate of the more labile blood components, such as granulocytes and platelets, after transplantation into adult mice has not been established. In the present work, the rate of replacement of the various hemopoietic cells in W/Wv transplant recipients was examined by exploiting host-donor differences in cellular markers (hemoglobin and glucose-phosphate isomerase). Limiting dilutions of +/+ parental cells were injected into F1-hybrid W/Wv anemic mice. A dose of 10(5) donor cells was necessary for the implantation of sufficient multipotent stem cells to alleviate the anemia. Erythrocyte parameters were not significantly different from normal values, and donor cells replaced at least 90% of the host erythrocytes by 20 weeks after injection. At this time, only 10% of the nucleated leukocytes and none of the platelets were donor-derived. The percentage of donor lymphocytes, granulocytes, and platelets increased slowly thereafter but did not completely replace the host elements by 41 weeks. The maintenance of host leukocytes during rapid donor-erythrocyte replacement indicates cell-specific amplification of committed progenitors.     

Regulation of hematopoiesis-IV: The role of interleukin-3 and bryostatin 1 in the growth of erythropoietic progenitors from normal and anemic W/Wv mice

We and others have established a role for T lymphocytes and their products in the regulation of erythropoiesis. Interleukin-3 (IL-3) is a multipotential lymphokine with burst-promoting activity that is produced by activated T lymphocytes. In the anemic, stem cell-defective W/Wv mouse we have described the absence of a functionally active thymocyte population that in normal animals enhances erythroid progenitor growth and stem cell self-renewal. In studies reported here we find that W/Wv mouse marrow responds to exogenous IL-3 by increased erythroid progenitor cell growth. The BFU-E and CFU-E from anemic donors are more sensitive to IL-3 than are those in +/+ marrow. We have recently observed a stimulatory effect of bryostatin 1 (a macrocyclic lactone derived from a marine invertebrate) on normal erythropoiesis in human bone marrow progenitor assays. To test the effects of this molecule on murine normal and anemic W/Wv cells we grew these cells in the presence of increasing doses of bryostatin 1. Bryostatin mimics the stimulatory action of IL-3 on W/Wv bone marrow. Polyclonal antibody directed against murine IL-3 blocks the stimulatory effect of bryostatin on erythropoiesis. Otherwise inactive thymocytes from W/Wv mice in coculture with W/Wv bone marrow showed stimulation of erythropoiesis in the presence of bryostatin. We believe that bryostatin may in part act by stimulating T lymphocytes to release physiologic concentrations of lymphokines.     

Purified murine hematopoietic stem cells function longer on nonirradiated W41/Wv than on +/+ irradiated stroma

Mouse bone marrow (BM) was separated into low-density, lineage- negative, wheat germ agglutinin-positive (WGA+), Rhodamine-123 bright (Rhbright) or dim (Rhdim) cells to obtain populations that were highly enriched for committed progenitors (Rhbright cells) or for more primitive stem cells (Rhdim). When 2,500 Rhbright or Rhdim cells were seeded onto 6-week-old irradiated (20 Gy) long-term BM cultures (LTBMC), the nonadherent cell production from Rhbright cells was transient and ended after 5 weeks. Production from Rhdim cells did not begin until week 3, peaked at week 5, and ended at week 8, when the irradiated stroma seemed to fail. Termination of cell production from Rhdim cells did not occur in nonirradiated LTBMC from W41/Wv mice. During peak nonadherent cell production, 25% to 30% of the cells in the nonirradiated LTBMC from W41/Wv mice had donor cell markers. Two approaches were tested to try to enhance the proportion or number of donor cells. Addition of Origen-HGF at the time of seeding Rhdim cells caused a nonspecific increase in both host and donor cell production, but a specific increase in production of donor cells was obtained by seeding the cultures at 2 weeks rather than 6 weeks. Limiting dilution of Rhdim cells gave the same frequency of wells producing cells on both irradiated +/+ and nonirradiated W41/Wv or W/Wv cultures.     

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.     

Latent deficiency of the hematopoietic microenvironment of aged mice as revealed in W/Wv mice given +/+ cells

The macrocytic anemia of W/Wv mice can be cured by injection of +/+ bone marrow cells (BMC) from WBB6F1 mice. However, it has been observed that some W/Wv recipients appear to "lose" their cure with time, an effect that does not appear to be related to the age of the BMC donor. The present study was undertaken to determine the effect of recipient age on W/Wv responses to BMC injection. The effect of aging on erythroid parameters was similar in untreated W/Wv mice and +/+ controls. In both genotypes, hematocrit (HCT) and red blood cell count (RBC) decreased, and the modal red blood cell size (peak) increased between 13 and 150 weeks of age. As anticipated, mean HCT and RBC values were lower and peak values higher in W/Wv mice compared to +/+ controls at every age. However, the rate of decrease in HCT and RBC with age was the same for both genotypes, suggesting that the age effect and W gene effect were independent. Peak values increased slightly more with age for W/Wv than for +/+ controls. When female W/Wv mice in three age groups (23.5, 70, and 91.5 weeks old) were injected with 5 x 10(5) BMC from 20-week-old +/+ female donors and HCT, RBC, and peak were determined monthly, improvement was seen in most W/Wv recipients. However, in the older mice this improvement was slower and often was not sustained; 100% of the youngest recipients, 80% of the middle-aged, and only 30% of the older groups were cured after 3 months. Taken together, these data suggest a latent deficiency of the aging hematopoietic microenvironment that is revealed in W/Wv mice by the stress of continuing erythroid demand on the limited number of normal donor BMC.     

Engraftment of bone marrow transplants in W anemic mice measured by electronic determination of the red blood cell size profile.

Defective stem cells of WBB6F1-W/Wv mice produce macrocytic red blood cells (RBCs); stem cells of WBB6F1-+/+ mice produce normocytic RBCs. Utilization of the Coulter counter channelyzer permitted good dissociation between the size distribution of populations of +/+ and W/Wv RBCs. Peaks (mean cell volumes) for +/+ and W/Wv RBCs have been determined to be between the 30th and 40th channel and 50th and 60th channel, respectively. Variability of profiles for individual mice of both genotypes did not exceed the variability of separate determinations of the same cell suspension from a single mouse. Admixture (approximately 15%) of either type of erythrocytes could be quantitatively detected by this method. One week after transplant of 10(7) +/+ marrow cells into W/Wv recipients, 25% of donor type erythrocytes were detected. Eighteen days post-graft, concentration of +/- normocytes exceeded the concentration of macrocytes in the W/Wv recipients' circulation. Approximately 45 days post-transplant, the proportion of macrocytes decreased below the 10% detectable level. Calculation of the daily RBC production rate during repopulation and estimation of the number of RBCs produced by a single hematopoietic colony were determined. The RBC size profile was found to be a convenient method for studying the effect of implantation of W/Wv marrow into lethally irradiated +/+ mice. This method proved suitable for repetitive determination of the size population in individual transplanted mice.     

Natural resistance of W/Wv mice to ethanol-induced gastric lesions and its abrogation by bone marrow grafting: possible role of mast cells and LTC4

The extent of ethanol-induced acute gastric lesions, gastric leukotriene C4 (LTC4) levels, and the number of gastric mucosal mast cells were examined in mast cell-deficient W/Wv mice, normal litter-mate +/+ mice, and bone marrow-reconstituted W/Wv mice. After administration of ethanol, +/+ mice developed gastric lesions and elevation of gastric LTC4 levels in a dose dependent manner. In mast cell-deficient W/Wv mice, the extent of gastric lesions was far less than that of +/+ mice and the level of gastric LTC4 was not significantly altered. This difference was not due to anemia because blood-transfused non-anemic W/Wv mice were still resistant to ethanol-induced gastric lesions. When W/Wv mice were reconstituted with +/+ bone marrow cells, their natural resistance against ethanol-induced gastric lesions was abrogated. The extent of gastric lesions of bone marrow-reconstituted W/Wv mice paralleled with the increase in number of gastric mucosal mast cells and also with the level of gastric LTC4. Furthermore, ethanol-induced gastric lesions in bone marrow-reconstituted W/Wv mice was inhibited by pretreatment with 5-lipoxygenase inhibitor, AA-861, in a dose dependent manner. These results suggest that LTC4 may, even if it is not a prerequisite factor for ethanol induced acute gastric lesions, act as the amplitier in the sequential events of the pathogenesis.     

Effect of anti-CSF on in vivo hemopoiesis

Studies were undertaken to determine whether colony-stimulating factor in the serum is important in the control of granulopoiesis and monocytopoiesis. Groups of mice were injected with either antiserum to colony-stimulating factor (CSF) or normal rabbit serum every 12 h for intervals of 6-7 days. Antibody treatment did not lead to a reduction in circulating granulocytes or monocytes nor a decrease in marrow cellularity. In further studies, two dose levels of purified anti-CSF were employed; rabbit IgG served as a control. Virtually no effect was observed on blood monocytes, blood granulocytes, or marrow granulocytes; however, antibody treatment did cause a decline in marrow colony-forming cells and a reciprocal increase in erythroid progenitor cells. Immunoassays showed a virtual absence of serum anti-CSF after five days, despite continued administration of the antibody. Modified immunoassays indicated that this was due to development of murine antibodies directed against anti-CSF. In further experiments, mice received the purified antibody or whole antiserum in conjunction with cortisone to reduce murine antibody formation. Despite persistent anti-CSF levels in the serum, blood granulocytes, blood monocytes, and marrow precursor cells were unaffected. These findings suggest that serum levels of CSF do not appear to be responsible for in vivo granulopoiesis or monocytopoiesis.     

Protection in lambs vaccinated with Haemonchus contortus antigens is age related, and correlates with IgE rather than IgG1 antibody

The involvement of mucosal mast cells (MMC) in protection against infection with the murine nematode parasite Trichuris muris was studied in genetically mast cell-deficient WBB6F1-W/Wv mice and their normal littermates WBB6F1-+/+ mice. Expulsion of T. muris worms occurred in infected +/+ mice, whereas no worm expulsion was observed in infected W/Wv mice where the infection persisted until at least day 46 postinfection. No MMC responses were induced in either infected W/Wv or +/+ mice. Specific IgG1and IgG2a antibodies to T. muris excretory/secretory antigens were observed in infected W/Wv and +/+ mice, and antibody production showed similar kinetics. Interleukin 4 production by concanavalin A (Con A)-stimulated mesenteric lymph node cells (MLNC) was induced preferentially in infected +/+ mice. T. muris infection increased the levels of IFN-gamma produced by Con A-stimulated MLNC of infected W/Wv and +/+ mice, with the levels of IFN-gamma in infected W/Wv mice being higher than those in infected +/+ mice. Taken together, these results indicate that W/Wv and +/+ mice are susceptible and resistant to T. muris infection, respectively, and that MMC responses are not required for protective immunity.     

Vascular endothelium as a regulator of granulopoiesis: production of colony-stimulating activity by cultured human endothelial cells

Colony-stimulating activity is a regulatory factor(s) that promotes differentiation of hemopoietic stem cells to mature granulocytes and macrophages; in man it has been found that blood monocytes, lymphocytes, and tissue macrophages produce it. In an effort to identify other potenitally physiologic tissue sources of colony- stimulating activity, we have studied the capacity of primary cultures of human vascular endothelial cells to produce colony-stimulating activity. Medium conditioned by incubation with endothelial cultures contained activity that promoted granulocyte-macrophage colony formation of nonadherent human and murine marrow cells. Exposure of endothelial cultures to 0.1-5.0 microgram/ml S. typhosa endotoxin for 6- 72 hr enhanced colony-stimulating activity production. Similarly, incubation of endothelial cells with lysates of human blood granulocytes, or cocultivation with intact granulocytes, resulted in increased colony-stimulating activity levels. In 7-14 day cultures, freshly isolated endothelial cells, incorporated into agar underlayers, consistently stimulated more colony formation by nonadherent human marrow cells than comparable numbers of blood monocytes. These data indicate that: (1) cultured human endothelial cells are a potent source of colony-stimulating activity; (2) they respond to endotoxin and granulocytes and their contents by producing increased amounts of CSA; and (3) they produce morea colony-stimulating activity, than human blood monocytes under standardized conditions in vitro. These observations suggest that the vascular endothelium may play a role in the physiologic regulation of granulopoiesis.     

AKR murine leukemia viruses and long-term bone marrow cultures from AKR and SJL mice

We have evaluated the effects of AKR mouse retroviruses on a system of long-term bone marrow cultures which allows prolonged replication of hemopoietic stem cells (CFUs) and granulocyte-monocyte progenitors with production of mature granulocytes. Ecotropic-nononcogenic virus is expressed in all cultures and does not adversely effect stem cell replication. We found, however, that cultures established from mice treated in vivo with lymphomagenic AKR viruses (Gross murine leukemia virus and AKR SL3 murine leukemia virus) showed a more rapid decline in CFUs and CFUc than cultures from "normal" AKR mice. Only the former cultures were found to produce lymphomagenic viruses. Furthermore, it was shown that addition of lymphomagenic virus to three-week marrow cultures established from AKR and SJL mice also caused a prompt decline in progenitor cell production and granulopoiesis when compared to medium-treated control cultures. The fate of all cultures was a loss of CFUc and granulocytes with a continuous production of macrophages. The change to macrophage production occurred earlier in the cultures treated with lymphomagenic virus or in those derived from lymphomagenic virus-treated animals. A study of these bone marrow-drived macrophage cultures, as well as cultures of similar morphology but of thymic origin, showed that they could be maintained as continuous lines. Cells from three of the cultures from lymphomagenic virus-treated animals plroduced locally growing sarcoma when inoculated into mice. The macrophages from the declined marrow cultures differed in certain properties from those of the established lines.     

Increased granulocytic, erythrocytic, and megakaryocytic progenitors in myelofibrosis with myeloid metaplasia

Nucleated cells obtained from blood and/or bone marrow of patients with myelofibrosis with myeloid metaplasia (MMM) were cultured in diffusion chambers (DC) implanted into the peritoneal cavities of irradiated mice. A total of five blood studies and two bone marrow studies were performed using cells obtained from five patients. The DC were harvested at intervals from the host mice and the total and differential cellularity of DC contents were evaluated. The results obtained from MMM cultures were compared with those from similar cultures of blood cells and marrow cells of four and six normal individuals respectively. The proliferation and maturation of the granulocytic, erythrocytic, and megakaryocytic lines in MMM cultures occurred in an orderly fashion as they occur in vivo. The patterns of proliferation and maturation of the three cell lines in cultures after day 7 suggest that they primarily originate from progenitor cells. The numbers of granulocytes in the multiplicative pool, recognizable red cell precursors, and megakaryocytes recovered were significantly greater from the MMM cultures than those from the normal blood or marrow cultures. These results suggest that the blood and marrow cells of MMM patients have increased numbers of progenitors for granulocytes, erythrocytes, and megakaryocytes.     

Bone Marrow Cell Culturing in Double Diffusion Chambers

A double diffusion chamber technique (DDC) has been established. The bone marrow cells (BMC) were cultured in the peritoneal cavity of mice in DDC consisting of 2 compartments separated from one another by a Millipore membrane. One chamber half contained the mouse bone marrow target cells, and the other half (regulator compartment) either medium (control), spleen cells or BMC. In the controls the BMC proliferated rapidly from day 2, and the cell yield on day 7 was reduced by only 20% when compared with single diffusion chambers. Diffusible factors from spleen cells stimulated the growth of CFU-S and CFU-C in the bone marrow, and increased the number of granulocytes and macrophages harvested in 7 day cultures. Conversely, BMC in the regulator compartment depressed granulopoiesis in the other chamber half.     

Phorbol 12-myristate 13-acetate-induced development of functionally active mast cells in W/Wv but not Sl/Sld genetically mast cell-deficient mice

The normal skin and other tissues of adult genetically mast cell-deficient WBB6F1-W/Wv or WCB6F1-Sl/Sld mice contain less than 1.0% the number of mast cells present in the corresponding tissues of the congenic normal (+/+) mice. We previously reported that mature dermal mast cells developed locally in the skin of W/Wv, but not Sl/Sld, mice at sites of chronic idiopathic dermatitis. We now report that the repeated application of phorbol 12-myristate 13-acetate (PMA) to the ear skin of either W/Wv or +/+ mice induces both dermatitis and a striking and dose-dependent increase in the number of dermal mast cells. The number of dermal mast cells at sites treated for 6 weeks with 5 micrograms PMA, three times per week, was 39 +/- 7/mm2 and 305 +/- 34/mm2 for W/Wv and +/+ mice, respectively; the corresponding values for vehicle-treated skin were 1.5 +/- 1.0/mm2 and 145 +/- 8/mm2, respectively. The PMA-induced dermal mast cells in W/Wv mice appeared mature by morphology, stained with the heparin-binding fluorescent dye, berberine sulfate, and were competent to express IgE-dependent passive cutaneous anaphylaxis responses. The development of mast cells was a local, not systemic, effect of PMA treatment. PMA treatment also induced dermatitis in both WCB6F1-Sl/Sld and +/+ mice, but was associated with increased numbers of dermal mast cells only in the WCB6F1(-)+/+ mice. PMA treatment had no detectable effect on the ability of bone marrow-derived cultured mast cells to survive in the skin of Sl/Sld mice. These findings establish a convenient model system for analyzing factors associated with the development of endogenous populations of mast cells in genetically mast cell-deficient W/Wv mice.     

Recombinant human erythropoietin has little influence on megakaryocytopoiesis in mice

The availability of a preparation of recombinant human erythropoietin (rEp) prompted us to investigate the role of Ep in the regulation of megakaryocytopoiesis in mice, using experimental procedures by which the effects on the mitotic and post-mitotic compartment of megakaryocytes could be evaluated separately. In agar cultures of murine bone marrow cells, either serum-depleted or serum-supplemented, rEp (0.1-2 U/ml) did not stimulate megakaryocyte colony formation and when it was added to suboptimal amount of spleen cell-conditioned medium (SCM), it failed to show a significant synergistic activity. On the contrary, rEp increased the number of megakaryocytic colonies developed from splenic precursors in the presence of suboptimal amounts of SCM, although it was unable per se to stimulate colony formation. The effects of rEps on megakaryocyte maturation and platelet production were studied in vivo evaluating the incorporation of 75Se-selenomethionine into platelets, the platelet count and platelet size, and the number of megakaryocyte precursors (small acetylcholinesterase positive cells, sAchE) in the bone marrow of mice injected with 1-8 U of rEp. No modification of these parameters was found in comparison with control mice. On the other hand, rEp increased the number of recognizable splenic megakaryocytes in a dose-dependent fashion. These data suggest that rEp has little influence on megakaryocytopoiesis, at least at the doses we used and which are known to elicit a maximal response of erythropoiesis. However, a subset of megakaryocytes with particular kinetic properties, such as those in the spleen of mice, may be responsive to relatively high doses of rEp. The significance of this observation in the overall regulation of megakaryocytopoiesis remains to be determined.