Hematopoietic stem cell function in motheaten mice

Mice homozygous for the autosomal recessive mutation "motheaten" have normal numbers of multipotential hematopoietic stem cells in the bone marrow and spleen as determined by spleen colony assay. Histologic examination shows no qualitative abnormality in morphology of stem cell colonies in recipients of bone marrow or spleen cells from motheaten mice. Despite the apparently normal ontogeny, distribution, and differentiative capacity of CFU stem cells, bone marrow and spleen cells from motheaten mice fail to save congenic +/+ lethally gamma-irradiated hosts. This impaired lifesparing capacity is not due to defective self-renewal but appears to be due in part to pulmonary hemorrhage from alveolar capillaries in the gamma-irradiated hosts. Treatment of motheaten mice with 500 R gamma-irradiation followed by reconstitution with normal bone marrow cells increases the lifespan of this mutant to 10 months of age. The early onset of pneumonitis and subsequent short lifespan of motheaten mice is determined at the level of progenitor cells in the bone marrow.     

Murine "viable motheaten" mutation reveals a gene critical to the development of both B and T lymphocytes

In lethally irradiated normal mice reconstituted with both normal and autoimmune mutant viable motheaten (mev) bone marrow, the mev-derived B and T cells display aberrant behavior, while those derived from the normal bone marrow develop and function normally. The observed developmental abnormalities of mev B and T lymphocytes are therefore intrinsic to these cell types, rather than being determined by defective influences from the cells' environment. These data bring into question the in vivo significance of reported intercellular regulatory defects in motheaten (me) and mev mice and suggest that these mutations affect a gene whose product acts cell autonomously in the development of several hematopoietic cell lineages including B and T lymphocytes.     

Evidence for migration of donor bone marrow stromal cells into recipient thymus after bone marrow transplantation plus bone grafts: A role of stromal cells in positive selection

Intrathymic T-cell differentiation is characterized by two selection events: positive and negative selection. It has been shown that thymic epithelial cells in the cortex are involved in the positive selection, while macrophages and dendritic cells, derived from hemopoietic stem cells, are involved in the negative selection. Here we investigate whether donor-derived bone marrow stromal cells can migrate into the thymus and participate there in positive selection after bone marrow transplantation plus bone grafts (to recruit bone marrow stromal cells).Allogeneic bone marrow transplantation with or without bone grafts was carried out in the [C57BL/6-->C3H] combination. Fluorescence-activated cell sorter analyses of recipient thymic adherent cells showed that donor-type bone marrow stromal cells exist in the thymus of mice that received bone marrow plus bone grafts but not in the mice that received bone marrow cells alone. Histological examination using confocal microscopy also confirmed the existence of donor-type stromal cells in the thymus of mice that received bone marrow cells plus bones. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice show donor-type major histocompatibility complex-restriction.These findings strongly suggest that stromal cells can migrate from the bone marrow to the thymus, where they participate in the positive selection of thymocytes.     

Hematologic abnormalities of the immunodeficient mouse mutant, viable motheaten (mev)

We have studied the hematopoietic system of the immunodeficient mouse mutant, viable motheaten (mev/mev). These mice usually die by 9 weeks of age from severe pneumonitis. The lungs at that time are infiltrated with granulocytes, macrophages, and lymphocytes. Granulocyte and macrophage precursor cells (CFU-GM) are dramatically increased in the spleens of mev/mev mice, whereas the bone marrow population of these precursors is decreased when compared with littermate control animals. The CFU-GM population retained its normal dependence on granulocyte-macrophage colony-stimulating factor (GM-CSF) for proliferation and differentiation. In contrast, the frequency of an erythroid precursor (CFU-E) was dramatically increased in spleen and showed increased sensitivity to erythropoietin (Epo). Moreover, a splenic CFU-E subpopulation formed normally appearing erythroid colonies in the absence of exogenous Epo. The bone marrow CFU-E population was significantly diminished in size when compared with either wildtype C57BL/6J mice or mice heterozygous for the mev allele. Unlike the CFU-E population, erythroid burst-forming unit (BFU-E) frequency in mev/mev mice was diminished both in bone marrow and in spleen, although the total number of splenic BFU-E was increased because of splenomegaly in these animals. BFU-E retained their dependence on the presence of both Epo and a source of interleukin 3 (IL-3) for proliferation and differentiation into erythroid bursts. Spleen cells from mev/mev mice, when stimulated in vitro with pokeweed mitogen, failed to produce significant quantities of IL-3. Comparison with medium or +/mev heterozygotes revealed that mev/mev spleen cell-conditioned medium showed a 40-fold reduction in burst-promoting activity. Thus, in viable motheaten mice, there is a major shift in hematopoiesis from bone marrow to spleen, which is accompanied by a diminished capacity of spleen cells to produce burst-promoting activity. These data and those from other studies suggest that the hematopoietic microenvironment of marrow may be impaired in this mutant.     

Effects of the motheaten gene on murine B-cell production

The rapidly fatal autoimmune disease in the mutant mouse known as motheaten is caused by an autosomal recessive gene and is characterized by hypergammaglobulinemia and autoantibody production, among other defects. The cellular kinetics of B-cell maturation were investigated in three-week-old motheaten mice and their normal littermates to determine whether any abnormality in cell production of the B lineage could be correlated with B-cell hyperactivity. The production rates and renewal times of newly produced bone marrow, splenic small B-lymphocytes, and splenic plasma cells were examined by in vivo tritiated-thymidine administration using a pulse-chase protocol and radioautography of immunofluorescence-stained cells. Because small B-lymphocytes in both organs were produced at comparable rates in the mutant mice and in their normal littermates, primary B-cell production was unaffected in the mutant mice. In contrast, splenic plasma cells were produced 10-30 times faster in motheaten mice than in normal mice. The enhanced rate of plasma cell production in motheaten mice could be correlated with a concurrent increased loss of labeled large B-lymphocytes, presumably rapidly dividing activated B cells. Thus, the excessive antibody production in motheaten mice may be reflected by the increased plasma cell production.     

The effects of allografts of thymic epithelial reticular cells on the lymphoid tissues of neonatally thymectomized mice

Neonatal thymus placed in a Millipore diffusion chamber for one weekloses its cortical cells, while the epithelial reticular cells remain viable.Grafts of these remnants in neonatally thymectomized mice are completely reconstituted from cells of the thymectomized host. Evidence ispresented which suggests that the bone marrow may be a source of cellswhich reform the thymus cortex.

These remnant grafts result in lymphoid reconstitution of neonatallythymectomized mice. The grafted animals also reject allografts of skin andlymphoma cells in a normal manner. However, these C3H mice, neonatallythymectomized and reconstituted with allografts of AKR thymic epithelialreticular cells, are tolerant to grafts of AKR lymphoma cells.

Submitted on February 23, 1966 Accepted on June 26, 1966     

Androgen receptors in thymic epithelium modulate thymus size and thymocyte development

Castration of normal male rodents results in significant enlargement of the thymus, and androgen replacement reverses these changes. Androgen-resistant testicular feminization (Tfm) mice also show significant thymus enlargement, which suggests that these changes are mediated by the androgen receptor (AR). The cellular targets of androgen action in the thymus are not known, but may include the lymphoid cells (thymocytes) as well as nonlymphoid epithelial cells, both of which have been believed to express AR. In the present study immunohistochemical analysis and hormone binding assays were used to demonstrate the presence of AR in thymic epithelial cells. The physiological significance of this epithelial cell AR expression was defined by further studies performed in vivo using chimeric mice, produced by bone marrow transplantation, in which AR expression was limited to either lymphoid or epithelial components of the thymus. Chimeric C57 mice engrafted with Tfm bone marrow cells (AR(+) epithelium and AR(-) thymocytes) had thymuses of normal size and showed the normal involutional response to androgens, whereas chimeric Tfm mice engrafted with C57 bone marrow cells (AR(-) epithelium and AR(+) thymocytes) showed thymus enlargement and androgen insensitivity. Furthermore, phenotypic analyses of lymphocytes in mice with AR(-) thymic epithelium showed abrogation of the normal responses to androgens. These data suggest that AR expressed by thymic epithelium are important modulators of thymocyte development.     

Stimulatory effects of products from inflammatory exudates upon stem cell production

The present study is based on the hypothesis that a humoral agent produced in inflammatory exudates is transported to the bone marrow where it stimulates CFU-S to differentiate into leukocytic precursors. Inflammatory exudates were produced by the subcutaneous implantation of a sterile acrylic cup filled with bacteria-free Hanks' balanced salt solution (HBSS) into B6D2F1 mice. The cups were removed 24 h later and the cells were separated from the supernatant. Cells collected from individual mice were pooled, as was the supernatant. Thereafter, normal mice received via tail vein either exudate cells, supernatant, plasma from cup-implanted or from normal mice, bone marrow cells or splenic cells from normal animals or HBSS. Another 24 h later bone marrow was removed from all mice and injected into previously lethally irradiated mice or suspended in vitro in agar-supported media. Nine days later the mice were euthanatized, the spleens removed, and colonies (CFU-S) counted. At the same time, colonies (CFU-C) were enumerated from the agar plates. The results clearly indicate that the bone marrow obtained from mice stimulated with either exudate cells or with super-natant or plasma obtained from cup-implanted mice contained a significantly greater number of CFU-S and CFU-C than did any one of the four control groups. These findings appear to substantiate the hypothesis.     

Augmentation of pre- and post-thymic T-lymphocyte responses in thymic epithelial cell grafted nude mice

Thymic epithelial (T-E) cell cultures from newborn mice, grown on dextran beads coated with denatured collagen, were injected subcutaneously into athymic "nude" mice. After 8 weeks, enhanced in vitro migration of T-E cell grafted nude mouse bone marrow cells occurred to thymus supernatants prepared from neonatal thymus cultures. Percent migration was equal to the migration of control AKR bone marrow. In vivo T-lymphocyte responses were also significantly enhanced in T-E cell grafted nude mice as compared to controls.     

Rationale for bone marrow transplantation in the treatment of autoimmune diseases.

Transplantation of normal bone marrow from C3H/HeN nu/nu (H-2k) mice into young MRL/MP-lpr/lpr (MRL/l; H-2k) mice (less than 1.5 mo) prevented the development of autoimmune diseases and characteristic thymic abnormalities in the recipient mice. When female MRL/1 (greater than 2 mo) or male BXSB (H-2b) mice (9 mo) with autoimmune diseases and lymphadenopathy were lethally irradiated and then reconstituted with allogeneic bone marrow cells from young BALB/c nu/nu (H-2d) mice (less than 2 mo), the recipients survived for more than 3 mo after the bone marrow transplantation and showed no graft-versus-host reaction. Histopathological study revealed that lymphadenopathy disappeared and that all evidence of autoimmune disease either was prevented from developing or was completely corrected even after its development in such mice. All abnormal T-cell functions were restored to normal. The newly developed T cells were found to be tolerant of both bone marrow donor-type (BALB/c) and host-type (MRL/1 or BXSB) major histocompatibility complex (MHC) determinants. Therefore, T-cell dysfunction in autoimmune-prone mice can be associated with both the involutionary changes that occur in the thymus of the autoimmune-prone mice and also to abnormalities that reside in the stem cells. However, normal stem cells from BALB/c nu/nu donors can differentiate into normal functional T cells even in mice whose thymus had undergone considerable involution, as in the case of BXSB or MRL/1 mice in the present studies. These findings suggest that marrow transplantation may be a strategy ultimately to be considered as an approach to treatment of life-threatening autoimmune diseases in humans. T-cell dysfunction in autoimmune-prone mice previously attributed to involutionary changes that occur in the thymus of these mice may instead be attributed to abnormalities that basically reside in the stem cells of the autoimmune-prone mice.     

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.     

Enhancement of the action of colony stimulating factor (CSF) by soluble component(s) of erythrocytes in mouse bone marrow cells cultures.

Rat erythrocytes, separated from other blood cells by SE-cellulose chromatography, were lysed by exposure to hypotonic solution, dialyzed and ultracentrifuged. The supernatant contained a substance which enhanced the activity of colony stimulating factor (CSF) in soft agar cultures of granulocyte-macrophage progenitor cells (CFU-C) from normal mouse bone marrow. The growth-enhancing effect of the erythrocyte factor was observed when mouse L-cell conditioned medium was used as the CSF source and also when serum from endotoxin-treated mice or from mice undergoing graft-versus-host reaction was used as the stimulant. The erythrocyte factor effect was also detected by 3H-thymidine uptake of bone marrow cells in liquid cultures. These results suggest that the effect of the erythrocyte factor on CSF is not restricted to CSF from a specific source nor to semi-solid agar cultures.     

Retrovirus-mediated gene transfer to purified hemopoietic stem cells with long-term lympho-myelopoietic repopulating ability.

Despite recent advances in marrow stem cell purification, controversy about the nature and heterogeneity of cells with the potential for long-term repopulation of lymphoid and myeloid tissues remains. Essential to the resolution of these questions is the use of strategies to track the progeny produced in vivo from individual hemopoietic stem cells in purified populations. We have used a procedure for obtaining highly enriched populations of stem cells with competitive repopulating ability from male mice (pretreated with 5-fluorouracil), and in this paper we present the results of studies in which small numbers (150-2000) of these cells were exposed to supernatant containing a helper-free recombinant retrovirus carrying the neomycin-resistance gene and then were transplanted together with 2 x 10(5) "compromised" female marrow cells into irradiated female recipients. Male cells--i.e., progeny of purified stem cells--were found in one or more of the tissues examined (peripheral blood, marrow, spleen, and thymus) in 28 of 28 mice evaluated at various times between 35 and 196 days after transplantation. In 20 of these mice (71%), the neomycin-resistance gene was also detected, although not always at a level that correlated with the proportion of male cells. Analysis of spleen colonies (day 12) generated in secondary recipients confirmed that viral integration was confined to male repopulating cells. In three mice direct evidence of a common clone in both lymphoid and myeloid tissues was also obtained. These results show the feasibility of retrovirus-mediated gene transfer to highly purified populations of lympho-myelopoietic stem cells with long-term (6 months) repopulating potential by using a supernatant infection protocol. This approach should facilitate further analysis of hemopoietic stem cell control in vivo and find future applications in the evolving use of bone marrow transplantation for hemopoietic rescue and gene therapy.     

Normal hematopoiesis is maintained by activated bone marrow CD4+ T cells

CD4(+) T cells produce hematopoietic-related cytokines and are essential for hematopoiesis stimulation during infection and hematologic recovery after bone marrow transplantation. However, it remains unclear if T cells are necessary to maintain normal hematopoiesis. We report here that, in T-cell-deficient mice, terminal differentiation of myeloid progenitors is defective, resulting in very low levels of granulocytes in the periphery. Hematopoiesis is restored after thymus graft or reconstitution with CD4(+) T cells but not CD8(+) T cells. Bone marrow CD4(+) T cells have an activated phenotype and produce cytokines, apparently, in the absence of exogenous stimulation. Transgenic mice carrying T-cell receptor specific for an ovalbumin peptide presented in the context of a specific class II molecule (I-A(d)) (DO11.10 RAG(-/-)) show the same hematopoietic deficiency as athymic mice. Their bone marrow CD4(+) T cells are not activated, suggesting that hematopoiesis maintenance requires the presence of cognate antigen in order to activate bone marrow T-helper cells. In fact, priming of transgenic mice with ovalbumin restores normal hematopoiesis. The data show that the current concept of "normal hematopoiesis" does not reflect a basal bone marrow activity, but it is an antigen-induced state maintained by constant activation of bone marrow CD4(+) T cells.     

Thymic control of proliferation of T cell precursors in bone marrow.

The activity of T lymphocyte precursors (pre-T cells) in the bone marrow of mice was measured by the concanavalin A response synergy assay. Pre-T cell levels were low in marrow of neonatally thymectomized mice and could be restored to control values by treatment in vivo with an extract of mouse thymus. Levels of activity were also low in aging mice and again could be restored by thymic extract treatment. The most profound fall with aging was in the proliferating pre-T cell compartment as detected by tritiated thymidine suicide; and this compartment was restored by thymic extract treatment. Irradiation to the thymus, with the bone marrow shielded, caused a fall in resting pre-T cells in the bone marrow and a concomitant rise in proliferating cells. These results are consistent with a model of control of pre-T cell maturation in which the thymus senses the number of developing lymphocytes within it and responds to a fall in this number by increasing production of hormone. The hormone acts on resting pre-T cells in the marrow, stimulating some of them to proliferate, leave the bone marrow, and repopulate the thymus.     

The Influence of Thymus Cells in Hemopoiesis: Stimulation of Hemopoietic Stem Cells in a Syngeneic, In Vivo, Situation

Using the spleen colony-forming techniqueto assay hemopoietic stem cells, (CFUs)experiments have been carried out to investigate the role of thymus cells in hemopoiesis. The experiments were carried outby injecting live or killed thymus cells intolethally-irradiated mice together withhemopoietic tissue containing CFUs. Theeffect of thymus cells on endogenouscolony formation in sublethally-irradiatedmice was also investigated. Colony formation by normal bone marrow or spleen isunaffected by the addition of thymus cells,but cell populations damaged by radiationall demonstrate increased colony numberswhen live thymus cells are injected within48 hr of initiating colony formation. Endogenous colony formation resulting from anx-ray dose of 475 red is increased 2.9times: exogenous colony formation frombone marrow or spleen populations whoseCFUs content has been reduced to approximately 20% of normal with 180-rad-rays is increased 2.1 times. Studiesinvestigating this enhancement demonstrate that at least some of the colonyforming cells require the cooperation oflive thymus cells. Under the conditions ofthese experiments, at least 10⁶ thymuscells must be injected to produce maximum enhancement.

Submitted on November 27, 1972 Revised on January 29, 1973 Accepted on February 16, 1973     

Novel B-cell maturation factor from spontaneously autoimmune viable motheaten mice.

Both in vivo and in vitro, mice homozygous for the viable motheaten mutation show severe immunodeficiency, polyclonal B-cell activation and Ig secretion, and spontaneous production of a lymphokine [B-cell maturation factor (BMF)] that directly drives the maturation of normal or tumor B cells to the state of active Ig secretion. BMF from motheaten mice is distinct from previously identified forms in its cells of origin (B cells) and biochemical characteristics (apparent Mr 15,000 by gel filtration and NaDodSO4/PAGE; pI 4.3 by chromatofocusing). Among the known murine single-gene models of autoimmunity, only motheaten mice show high levels of spontaneous BMF production, which therefore may be an important component in the development of this form of autoimmunity/immunodeficiency disease. The coincidence of spontaneous BMF production and uncontrolled Ig secretion within the same mutant mouse constitutes the strongest evidence to date for a significant physiological (in vivo) role for BMFs.     

Cellular Sites of Immunologic Unresponsiveness

The reconstitution of the immune response of lethally irradiated mice to human gamma-globulin is dependent on the synergistic action of bone marrow with thymus cells. Immunologic unresponsiveness appears to involve a functional defect at each of these cellular levels, inasmuch as neither bone marrow nor thymus cells from unresponsive donors are capable of demonstrating synergism in combination with their normal counterpart.     

A new pre-irradiation conditioning regimen which protects against radiation injury and facilitates engraftment of xenogeneic bone marrow.

Administration of a large number of xenogeneic (rat) bone marrow cells before lethal, total body irradiation in mice (preconditioning regimen) results in powerful protection from radiation damage, remarkable prolongation of their survival, facilitation of marrow engraftment and induction of rat chimerism. This protective effect is not exerted by thymus or spleen cells and does not depend on specific immune mechanisms. The preconditioning regimen is more efficient when the bone marrow cells are viable and when the cells are inoculated intravenously a few hours before irradiation.     

Enhancement of hematopoiesis and lymphopoiesis in diet-induced obese mice

A rodent model of diet-induced obesity revealed that obesity significantly altered hematopoietic and lymphopoietic functions in the bone marrow and thymus. C57BL/6 mice were fed a mixed high-fat diet (HFD) of 45% fat or 10% fat diet (lean controls) for 180 d. A sustained increase in the numbers of cells found in bone marrow and thymus of HFD mice occurred from day 90 to day 180. However, with the exception of a 10-18% increase in the proportion of lymphocytes, the composition of monocytes, granulocytes, erythrocytes, and mixed progenitor lineages remained normal in the marrow. Likewise, thymuses of HFD mice increased 30-50% in size compared with controls, with analogous increases in thymocyte numbers. The overall thymus cellular composition remained normal. Although increased blood and lymphatic volume in obese mice would play a role in increased hematopoiesis, there were large and disproportionate increases in blood leukocytes of HFD mice, indicating that homeostasis was not maintained. Leptin, which promotes lymphopoiesis and myelopoiesis, reached 100 ng/mL in sera from HFD mice. Moreover, a three- to sixfold increase in adipocytes in marrow resulted in spiked leptin mRNA expression in bones of HFD mice compared with lean controls. Other cytokines and growth factors did not show any increases in obese marrow. The substantial increase in lymphopoietic and hematopoietic processes in HFD mice indicates that the primary tissues are another facet of the immune system dysregulated by obesity, which was perhaps fostered by higher amounts of leptin in marrow and serum.