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.     

The decrease in long-term marrow repopulating capacity seen after transplantation is not the result of irradiation-induced stromal injury

Marrow cells from nonirradiated F1-W/Wv mice repopulated slightly less well than cells from lethally irradiated recipients. Therefore, avoiding irradiation of recipients did not improve the relative repopulating ability of their marrow cells. In other experiments, F1-W/Wv mice were transplanted by parabiosis with marrow of WBB6F1-+/+ (F1-+/+) mice, avoiding cellular handling and irradiation. Marrow cells transplanted to F1-W/Wv mice by this procedure demonstrated slightly better repopulating ability than did marrow cells transplanted by injection. However, they performed no better than those transplanted by parabiosis to irradiated F1-+/+ recipients. Significant impairment of stromal function after irradiation was not indicated. Apparently, stem cell damage caused by transplantation may have greater importance in causing loss of stem cell replicative potential than effects of irradiation-induced stromal injury.     

Different repopulation profile between erythroid and nonerythroid progenitor cells in genetically anemic W/Wv mice after bone marrow transplantation

Repopulation kinetics of erythrocytes and neutrophils and replacement of hematopoietic progenitors were studied in genetically anemic (WB x C57BL/6)F1-W/Wv (WBB6F1-W/Wv) hosts after bone marrow transplantation from C57BL/6-bgJ/bgJ or C57BL/6-bgJ/bgJ;Pgk-1a/Y mice. Electrophoretic pattern of hemoglobin was used as a marker of donor-type erythrocytes, giant granules of bgJ/bgJ mice as a marker of donor-type neutrophils, and A-type phosphoglycerate kinase-1 (PGK-1) as a marker of hematopoietic colonies produced by donor-derived progenitor cells. Repopulation of donor-type erythrocytes was significantly faster than that of donor-type neutrophils. Moreover, the extent of replacement was greater for erythroid progenitor cells than for nonerythroid progenitor cells. When nonirradiated WBB6F1-W/Wv mice with B-type PGK-1 received 10(5) bone marrow cells from C57BL/6-bgJ/bgJ;Pgk-1a donors, only approximately 20% replacement of erythroid progenitor cells gave rise to total reconstitution of erythrocytes. The present result suggests that normal multipotential stem cells may preferentially differentiate into erythroid lineage cells in anemic WBB6F1-W/Wv hosts and that normal erythroid progenitor cells may suppress the differentiation of erythroid progenitors of WBB6F1-W/Wv hosts.     

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.     

Lymphoid differentiation of the hematopoietic stem cell that reconstitutes total erythropoiesis of a genetically anemic W/Wv mouse

We investigated whether the stem cell that reconstitutes total erythropoiesis of a WBB6F1-W/Wv mouse differentiates into lymphoid lineage. The electrophoretic pattern of hemoglobin was used as a marker of the reconstitution; 3-phosphoglycerate kinase (PGK), an X chromosome-linked enzyme was used as a tool for estimating clonality. We injected 10(5) bone marrow cells of 5-FU treated C57BL/6-Pgk-1b/Pgk-1a female mice, in which each stem cell had either A-type PGK or B-type PGK due to random inactivation of one of two X chromosomes, into genetically anemic (WB x C57BL/6)F1-W/Wv (hereafter WBB6F1-W/Wv) mice that contained only B-type PGK. The recipient WBB6F1-W/Wv mice, in which erythropoiesis was reconstituted with donor cells for a long term, were killed and the PGK patterns of bone marrows, thymus, lymph nodes, and Peyer's patches were examined. A considerable amount of A-type PGK was detected in the lymphoid organs of the WBB6F1-W/Wv mice in which erythrocytes showed only A-type PGK when killed. In contrast, A-type PGK was scarcely detectable in the lymphoid organs of the WBB6F1-W/Wv mice in which erythrocytes showed only B-type PGK when killed. The present results suggest that the hematopoietic stem cells estimated by the erythropoiesis reconstituting assay differentiate into lymphoid lineage and that the long-term erythropoiesis reconstitution assay is useful for detecting the true primitive hematopoietic stem cells.     

Hybrid resistance to parental bone marrow-derived cultured mast cells in the skin but not in the peritoneal cavity of (WB X C57BL/6)F1-W/Wv mice

When cultured mast cells of (WB X C57BL/6)F1-+/+(WBB6F1-+/+) and WB-+/+(WB) mice were directly injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, mast cell clusters appeared at the injection sites. Although in vitro colony-forming ability is comparable between cultured mast cells of WB mice and those of WBB6F1-+/+ mice, the number of WB mast cells necessary for the appearance of mast cell clusters in the skin of WBB6F1-W/Wv mice was significantly larger than the number of WBB6F1-+/+ mast cells. In spite of the presence of such an apparent hybrid resistance in the skin of WBB6F1-W/Wv mice to mast cells of the WB parent, both WB and WBB6F1-+/+ mast cells grow in the peritoneal cavity of WBB6F1-W/Wv mice with comparable efficiency. This is a demonstration of the tissue-related (nonrecirculating) expression of hybrid resistance against nonmalignant hematopoietic cells.     

Development of large numbers of mast cells at sites of idiopathic chronic dermatitis in genetically mast cell-deficient WBB6F1-W/Wv mice

The normal skin and other tissues of adult 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. As a result, genetically mast cell-deficient WBB6F1-W/Wv or WCB6F1-Sl/Sld mice are widely used for studies of mast cell differentiation and function. We found that mast cells developed at sites of idiopathic chronic dermatitis in WBB6F1-W/Wv mice and that the number of mast cells present in the skin of WBB6F1-W/Wv mice was proportional to the severity of the dermatitis (in ear skin, there were 33 +/- 4 mast cells/mm2 of dermis at sites of severe dermatitis v 9 +/- 3 at sites of mild dermatitis, 0.8 +/- 0.3 in skin without dermatitis, and 100 +/- 7 in the normal skin of congenic WBB6F1-+/+ mice; in back skin, the corresponding values were 2.0 +/- 0.6, 1.1 +/- 0.9, 0.025 +/- 0.025, and 26.2 +/- 3.2). The development of mast cells was a local, not systemic, consequence of the dermatitis. Thus, WBB6F1-W/Wv mice with severe dermatitis lacked mast cells in skin not showing signs of dermatitis and also in the peritoneal cavity, stomach, cecum, and tongue. Idiopathic chronic dermatitis was not associated with the local development of mast cells in WCB6F1-Sl/Sld mice, a mutant whose mast cell deficiency is due to a mechanism distinct from that of WBB6F1-W/Wv mice. These findings may have implications for understanding the nature of the mast cell deficiency in WBB6F1-W/Wv and WCB6F1-Sl/Sld mice and for the use of these mutants to analyze mast cell differentiation and function.     

Effect of 5-fluorouracil on "primitive" hematopoietic stem cells that reconstitute whole erythropoiesis of genetically anemic W/Wv mice

The potential to reconstitute the whole erythropoiesis of a genetically anemic (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mouse for at least 8 weeks was compared between 5-fluorouracil (5FU)-treated and nontreated bone marrow cells. C57BL/6-Pgk-1b/Pgk-1a female mice, in which each stem cell had either A-type or B-type phosphoglycerate kinase (PGK) owing to the random inactivation of one of two X chromosomes, were used as donors. As a marker of the reconstitution, electrophoretic pattern of hemoglobin was used. The concentration of the stem cells that reconstitute the whole erythropoiesis of WBB6F1-W/Wv mouse was higher in the marrow of donors that had received an injection of 5FU two days previously (two-day 5FU-treated) than in the marrow of nontreated donors. In the marrow of four-day 5FU-treated mice, however, the concentration was comparable to that of nontreated mice. The PGK electrophoretic pattern of WBB6F1-W/Wv mice reconstituted by nontreated marrow cells was comparable to the PGK pattern of WBB6F1-W/Wv mice reconstituted by four-day 5FU-treated marrow cells. Thus, a single stem cell with extensive proliferative potential rather than multiple spleen colony-forming units appeared to be responsible for the erythropoietic reconstitution in the transplantation of nontreated healthy marrow cells as well as 5FU-treated marrow cells.     

Bone marrow origin of mast cell precursors in mesenteric lymph nodes of mice

Concentration of mast-cell precursors in the mesenteric lymph node of (WB X C57BL/6)F1 hybrid mice (WBB6F1) were evaluated by a limiting dilution method. Cells from WBB6F1-+/+ mice were injected directly into the skin of WBB6F1-W/Wv mice which genetically lack tissue mast cells. Concentrations of mast-cell precursors were calculated from the proportion of injection sites at which mast cells appeared. Although immunization with horse serum significantly increased the concentration of mast-cell precursors in the mesenteric lymph node, the concentration in the lymph node remained about 10% that observed in the peripheral blood mononuclear cells. Since the bone marrow origin of mast-cell precursors in the mesenteric lymph node was demonstrated by using giant granules of beige (C57BL/6-bgj/bgj) mice as a marker, the immunization seemed to increase the migration of bone-marrow-derived mast-cell precursors from the peripheral blood to lymph node.     

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.     

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.     

Marrow transplantation in the treatment of a murine heritable hemolytic anemia

Mice with hemolytic anemia, sphha/sphha, have extremely fragile RBCs with a lifespan of approximately one day. Neither splenectomy nor simple transplantation of normal marrow after lethal irradiation cures the anemia but instead causes rapid deterioration and death of the mutant unless additional prophylactic procedures are used. In this report, we show that normal marrow transplantation preceded by sublethal irradiation increases but does not normalize RBC count. The mutant RBCs but not all the WBCs are replaced by donor cells. Splenectomy of the improved recipient causes a dramatic decrease in RBC count, indicating that the mutant spleen is a site of donor-origin erythropoiesis as well as of RBC destruction. Injections of iron dextran did not improve RBC counts. Transplantation of primary recipient marrow cells into a secondary host with a heritable stem cell deficiency (W/Wv) corrects the defect caused by residence of the normal cells in the sphha/sphha host. The original +/+ donor cells replace the RBCs of the secondary host, and the RBC count is normalized. Results indicate that the environment in the sphha/sphha host is detrimental to normal (as well as mutant) erythroid cells but the restriction is not transmitted.     

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.     

Intraperitoneally injected cultured mast cells suppress recruitment and differentiation of bone marrow-derived mast cell precursors in the peritoneal cavity of W/Wv mice

Bone marrow-derived mast cell precursors form large mast cell colonies in methylcellulose and are designated as L-CFU-Mast. The effect of differentiated mast cells on recruitment and differentiation of L-CFU-Mast was investigated by using genetically mast cell-deficient WBB6F1-W/Wv mice. Giant granules of C57BL/6-bgJ/bgJ (Chediak-Higashi syndrome) mice were used as a marker to identify the origin of L-CFU-Mast and differentiated mast cells. Practically no L-CFU-Mast are present in the peritoneal cavity of WBB6F1-W/Wv mice. When bone marrow cells of WBB6F1(-)+/+ mice were i.v. injected, the concentration of +/+(-)type L-CFU-Mast increased in the peritoneal cavity of WBB6F1-W/Wv mice and became several times greater than that of nontreated WBB6F1(-)+/+ mice. This increase of L-CFU-Mast was suppressed by a prior i.p. injection of bgJ/bgJ-type cultured mast cells. The differentiation of the +/+(-)type L-CFU-Mast to morphologically identifiable mast cells was also suppressed by the i.p. injection of bgJ/bgJ-type cultured mast cells. The present results suggest that the suppression of recruitment and differentiation of L-CFU-Mast is a physiological function of differentiated mast cells.     

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.     

Regulation of mast cell differentiation studied using the diffusion chamber technique

A homogeneous population of mast cells was obtained by culturing bone marrow cells of WBB6F1(-)+/+ mice. The proliferation of the cultured mast cells in diffusion chambers was investigated to examine whether the diffusion chamber technique was applicable for study of the regulation of mast cell proliferation. WBB6F1-W/Wv mice are genetically deficient in mast cells. When cultured mast cells of WBB6F1(-)+/+ mouse origin were directly injected into the peritoneal cavity of WBB6F1-W/Wv mice, the mast cells survived. In contrast, WBB6F1(-)+/+ mouse-derived cultured mast cells did not survive in diffusion chambers implanted in the peritoneal cavity of either WBB6F1-W/Wv or WBB6F1(-)+/+ mice. Because the coinoculation of NIH/3T3 cells supported the proliferation of mast cells in diffusion chambers, a certain type of cells in the peritoneal cavity appeared to have the same mast cell-supporting activity as NIH/3T3 cells. The magnitude of either interleukin 3-dependent or NIH/3T3 cell-dependent proliferation of mast cells in diffusion chambers was not significantly influenced by the genotype of chambers recipients (i.e., WBB6F1(-)+/+ or WBB6F1-W/Wv mice), suggesting that the previously reported inhibitory effect of mast cells on differentiation of mast cells may be mediated by direct contact between mast cells.     

Animal model of sclerotic skin. II. Bleomycin induced scleroderma in genetically mast cell deficient WBB6F1-W/W(V) mice

OBJECTIVE: Previously, we established a mouse model of scleroderma induced by repeated subcutaneous bleomycin injections. In this model, increased numbers of mast cells were observed in the lesional skin of dermal sclerosis, and degranulation of mast cells was prominent prior to the increase of mast cell numbers. Mast cells have been suggested to play an important role in tissue fibrosis. In this study, we investigated whether dermal sclerosis is also induced by bleomycin administration in genetically mast cell deficient WBB6F1-W/W(V) mice. METHODS: Bleomycin was subcutaneously injected every day in WBB6F1-W/W(V) and their normal littermate WBB6F1-+/+ mice for 4 weeks, and mice were analyzed for histological sclerosis, mast cell number, plasma histamine level, and hydroxyproline content. RESULTS: Four weeks' injections of bleomycin effected histological dermal sclerosis in both mast cell deficient and control strains; however, at 1 week, dermal sclerosis was induced only in WBB6F1-+/+ mice. Mast cells gradually increased in number around or on the edge of sclerotic lesions in WBB6F1-+/+ mice, as the dermal sclerosis developed. Hydroxyproline content of the skin of WBB6F1-+/+ mice was higher than that of WBB6F1-W/Wv mice at 1 week, but was not statistically significant. After 2 weeks' treatment with bleomycin, the hydroxyproline content of the skin was similar in both strains. The number of infiltrating macrophages and CD4+ T cells also gradually increased in both strains; however, the difference did not reach significance during the course of bleomycin treatment. CONCLUSION: These results show that mast cell is not necessary for inducing dermal sclerosis by bleomycin, and other types of inflammatory cells such as infiltrating macrophages or T lymphocytes may play a role in triggering induction of dermal sclerosis via fibrogenic cytokines. However, mast cell releasing mediators or cytokines may play a role in accelerating formation of dermal sclerosis, in particular, at an early phase of the sclerotic process, and not merely as a result of sclerosis.     

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.     

Megakaryocytopoiesis and granulopoiesis of W/Wv mice studied in long- term bone marrow cultures

Megakaryocytopoiesis and granulopoiesis of marrow cells from W/Wv mice were studied using a continuous liquid marrow culture system. Cells in the suspension phase were assayed weekly over a 16-week period for total nucleated cells, megakaryocytes, granulocytes, megakaryocytes and granulocyte-macrophage progenitor cells (CFU-Ms, CFU-GMs), and spleen colony-forming cells (CFU-Ss). Without hydrocortisone supplementation, proliferation of megakaryocytes, granulocytes, and their progenitor cells was significantly less in W/Wv cultures than in +/+ cultures. These cells became undetectable in both W/Wv and +/+ cultures at seven to 11 weeks in culture, after which only monocytes and macrophages proliferated in the cultures. Treatment of cultures with hydrocortisone improved megakaryocytopoiesis and granulopoiesis in both W/Wv and +/+ cultures. Following an initial lag phase of three to four weeks, proliferation of megakaryocytes, granulocytes, and their progenitor cells in W/Wv cultures equalled that observed in +/+ cultures and was sustained for 16 to 24 weeks. This improvement was associated with a sustained reduction in monocytes and macrophages. Despite improvements in megakaryocytopoiesis and granulopoiesis, production of macroscopic and microscopic spleen colonies by cells from W/Wv cultures remained severely reduced or absent. Studies of DNA synthesis rates of fresh marrow cells indicated that significantly fewer CFU-Ms and CFU-GMs were in cycle in W/Wv mice compared with +/+ mice. However, in hydrocortisone-treated W/Wv cultures, DNA synthesis rates of CFU-Ms and CFU-GMs increased markedly and equalled those observed for +/+ cultures. These results suggest that the improvements in megakaryocytopoiesis and granulopoiesis in hydrocortisone-treated liquid cultures is associated with a reduction in monocytes and macrophages and that progenitor cells of W/Wv mice have a proliferative defect that is correctable by hydrocortisone treatment in vitro.     

Partitioning of bone marrow into stem cell regulatory domains.

To examine the hypothesis that bone marrow consists of discrete stem cell regulatory volumes or domains, we studied spleen colony-forming unit (CFU-S) population growth kinetics in unirradiated WBB6F1-W/Wv mice receiving various doses of +/+ bone marrow cells. Assay of femoral marrow CFU-S content in the eight recipient dose groups revealed a family of growth curves having an initial dose-independent exponential phase and a subsequent dose-dependent deceleration phase. CFU-S content at the growth transition (inflection point) was not a simple linear function of inoculum dose but was shown rather to reflect a random distribution of initially seeded donor CFU-S in discrete volumes of recipient bone marrow. The inoculum dose resulting in a mean of 1 CFU-S per bone marrow sampling unit was estimated to be 17 x 10(6) bone marrow cells, corresponding to a total marrow uptake of approximately 5100 CFU-S (based on a seeding efficiency factor of 10%). If we assume single-hit kinetics, it follows that the recipient W/Wv bone marrow may contain approximately 5100 domains in which stem cell proliferation is geared to the density of the stem cell population. When the various inocula were corrected for multiple seeding in a given domain, the mean inflection point per domain was similar and indicative of five or so divisions before departure from exponential growth at approximately 20% of final CFU-S content 8 days after bone marrow injection. The partitioning of bone marrow into highly localized functional units is consistent with the putative regulatory role of short-range interactions between stem cells and essential stromal elements.