Generation of functionally distinct B lymphocytes from common myeloid progenitors.

Current models of adult haematopoiesis propose that haematopoietic stem cells (HSCs) differentiate into common lymphoid (CLP) and common myeloid (CMP) progenitors and establish an early separation between myeloid and lymphoid lineages. Nevertheless, the developmental potential of CMP-associated B cells suggests the existence of alternate pathways for B lymphopoesis. The aim of this study was to compare the developmental and functional properties of CMP- and CLP-derived B cells. While both populations matured through pro-B cell and transitional B cell intermediates in the bone marrow and spleen, respectively, following transfer into irradiated mice, mature CMP- and CLP-derived B cells exhibit distinct functional responses. Specifically, CMP-derived B cells did not respond to mitogenic stimulation to the same degree as their CLP-derived counterparts and secrete lower levels of IgM and the inflammatory cytokines such as interleukin (IL)-6 and IL-10. Together, these data suggest the existence of multiple pathways for generating functionally distinct B cells from bone marrow precursors.

Regulation of hematopoiesis by gap junction-mediated intercellular communication.

Gap junctions are intercellular channels formed by individual structural units known as connexins (Cx) that allow the intercellular exchange of small molecules between cells. The presence of Cx protein in bone marrow and thymic stromal cells and the demonstration that these cells are functionally coupled have led to the hypothesis that groups of stromal cells in the bone marrow and thymus form a functional syncytium through which their hematopoietic support capacity is coordinated. The validity of this hypothesis was recently tested in a newly developed strain of mice in which the gene encoding Cx43, the principal Cx expressed in hematopoietic tissues, was disrupted. Studies of myelopoiesis and lymphopoiesis in these Cx43-deficient mice revealed that expression of Cx43 in the bone marrow and thymus is critically important during periods of active hematopoiesis, such as during embryogenesis and after recovery from cytoablative treatments. The clinical implications of these observations, as well as issues that remain to be addressed to understand the mechanism(s) by which gap junctions regulate hematopoiesis, are addressed.

B cells are selectively associated with thymic cortical but not medullary pathology in NZB mice.

Abnormal expansion of autoantibody-synthesizing B cells and self-reactive T cells, which most likely escape negative selection within the thymus, have both been characterized and reasoned to play a role in the pathogenesis of autoimmunity in NZB mice. Support for this thesis has been our observation that NZB mice have severe cortical and medullary thymic microarchitectural defects. As a means to dissect the roles of T and B cells in the induction of such abnormalities, B cell-deficient NZB mice were bred by backcrossing the Igh6(null)allele on to the NZB background (NZB-muMT mice). Such mice showed undetectable levels of autoantibodies. NZB-muMT mice, as compared to wild-type NZB mice, had lower absolute numbers of CD4(+)T cells. Furthermore, thymic abnormalities in NZB-muMT mice were restricted to the medulla. These data suggest that, while B cells may play a role in thymic cortical abnormalities, the medullary abnormalities are induced by other mechanisms.

Increased expression of mXBP-1 (TREB-5) in thymic B cells in New Zealand mice.

New Zealand Black mice as well as several other murine models of murine lupus are well known for premature degeneration of thymus and development of autoimmunity. To focus on molecular events unique to murine lupus, we performed differential display using arbitrary primer pairs to distinguish NZB versus BALB/c thymus at 5 weeks of age. Following an extensive analysis of DNA bands that were either consistently up or downregulated and from studies of expression pattern of thymic genes by in situ nucleic acid hybridization, we focused on one clone that was consistently differentially expressed between NZB and BALB/c thymus. This clone was isolated, sequenced, and identified as the murine homologue of the human X box binding protein (hXBP-1), also known as TREB 5. mXBP-1 was found to be consistently upregulated in B cells in the thymic cortex of NZB and (NZBxNZW)F1, but not BALB/c, C3H/HeJ or C57BL/6 mice. In addition, it was dramatically elevated in MRL/ lpr but not MRL/++ mice; similarly, it was increased in BXSB/ Yaa male but not BXSB female thymic cortex. Of particular interest was an absence of mXBP-1 expression in the thymus of NZB/ Bln- Igh6(null)homozygotes. mXBP-1 has several putative functions, including the regulation of MHC class II expression and by virtue of its ability to recognize CRE-like elements shown to be involved in HTLV-1 transcription.

Bipotential B-macrophage progenitors are present in adult bone marrow.

According to the current model of adult hematopoiesis, differentiation of pluripotential hematopoietic stem cells into common myeloid- and lymphoid-committed progenitors establishes an early separation between the myeloid and lymphoid lineages. This report describes a rare and previously unidentified CD45R-CD19+ B cell progenitor population in postnatal bone marrow that can also generate macrophages. In addition to the definition of this B-lineage intermediate, the data indicate that a developmental relationship between the B and macrophage lineages is retained during postnatal hematopoiesis.

Anterior pituitary hormones, stress, and immune system homeostasis.

An extensive, and controversial, literature concluding that prolactin (PRL), growth hormone (GH), insulin-like growth factor-I (IGF-I), and thyroid hormones are critical immunoregulatory factors has accumulated. However, recent studies of mice deficient in the production of these hormones or expression of their receptors indicate that there are only a few instances in which these hormones are required for lymphocyte development or antigen responsiveness. Instead, a case is made that their primary role is to counteract the effects of negative immunoregulatory factors, such as glucocorticoids, which are produced when the organism is subjected to major stressors. The immunoprotective actions of PRL, GH, IGF-I, and/or thyroid hormones in these instances may ensure immune system homeostasis and reduce the susceptibility to stress-induced disease. These immuno-enhancing effects could be exploited clinically in instances where the immune system is depressed due to illness or various treatment regimens.

Helper (CD4(+)) and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin-deficient muscle.

Duchenne muscular dystrophy (DMD) and mdx mouse dystrophy result from mutations in the dystrophin gene. Although these mutations are primarily responsible for the defects that underlie the pathology of dystrophinopathies, other factors may contribute importantly to the pathology. In the present investigation, we tested whether T cells present in mdx muscles are activated and contribute significantly to the pathological process. Flow cytometric analyses showed a significantly higher frequency of activated CD44(high) T cells in the blood and muscle of mdx mice when compared to normal B10 mice. However, the frequency of activated T cells was not elevated in mdx lymph nodes, suggesting muscle-specific T cell activation. In vivo antibody-mediated depletions of CD4(+) T cells from mdx mice significantly reduced the amount of histologically discernible muscle pathology by 61% in mdx mice, while depletion of CD8(+) T cells resulted in a 75% decrease in pathology. Finally, adoptive transfer of mdx immune cells in combination with muscle extracts resulted in muscle pathology in healthy murine recipients. These results indicate that T cells promote the mdx pathology and suggest that immune-based therapies may provide benefit to DMD patients.

Humoral and cell-mediated immunity in mice with genetic deficiencies of prolactin, growth hormone, insulin-like growth factor-I, and thyroid hormone.

Prolactin (PRL), growth hormone (GH), insulin-like growth factor-I (IGF-I), and thyroid hormones have been proposed as critical immunoregulatory mediators, and their clinical use is being considered. The precise role played by each of these hormones in the generation of humoral and cell-mediated immune responses was assessed in a panel of mice with mutations that result in a selective reduction of PRL, GH, IGF-I, and/or thyroid hormone production. A surprising result, in view of previous studies indicating an immunoregulatory role for these hormones, was that all mice generated normal humoral and cell-mediated immune responses following challenge with T-independent and T-dependent antigens and with Listeria monocytogenes. A review of these findings in the context of previous data has resulted in the formulation of a working hypothesis proposing that these hormones act as anabolic and/or stress modulating mediators with effects on most cells, including those of the immune system. When considered in this context, it is possible to reconcile the contradictory data.

Expression of connexin 43 (Cx43) is critical for normal hematopoiesis.

Gap junctions are intercellular channels, formed by individual structural units known as connexins (Cx), that allow the intercellular exchange of various messenger molecules. The finding that numbers of Cx43-type gap junctions in bone marrow are elevated during establishment and regeneration of the hematopoietic system has led to the hypothesis that expression of Cx43 is critical during the initiation of blood cell formation. To test this hypothesis, lymphoid and myeloid development were examined in mice with a targeted disruption of the gene encoding Cx43. Because Cx43-/- mice die perinatally, initial analyses were performed on Cx43-/-, Cx43+/-, and Cx43+/+ embryos and newborns. The data indicate that lack of Cx43 expression during embryogenesis compromises the terminal stages of primary T and B lymphopoiesis. Cx43-/- embryos and neonates had a reduced frequency of CD4(+) and T-cell receptor-expressing thymocytes and surface IgM(+) cells compared to their Cx43+/+ littermates. Surprisingly, Cx43+/- embryos/neonates also showed defects in B- and T-cell development similar to those observed in Cx43-/- littermates, but their hematopoietic system was normal at 4 weeks of age. However, the regeneration of lymphoid and myeloid cells was severely impaired in the Cx43+/- mice after cytoablative treatment. Taken together, these data indicate that loss of a single Cx43 allele can affect blood cell formation. Finally, the results of reciprocal bone marrow transplants between Cx43+/+ and Cx43+/- mice and examination of hematopoietic progenitors and stromal cells in vitro indicates that the primary effects of Cx43 are mediated through its expression in the hematopoietic microenvironment.

The roles of prolactin, growth hormone, insulin-like growth factor-I, and thyroid hormones in lymphocyte development and function: insights from genetic models of hormone and hormone receptor deficiency.

An extensive literature suggesting that PRL, GH, IGF-I, and thyroid hormones play an important role in immunity has evolved. Because the use of one or more of these hormones as immunostimulants in humans is being considered, it is of critical importance to resolve their precise role in immunity. This review addresses new experimental evidence from analysis of lymphocyte development and function in mice with genetic defects in expression of these hormones or their receptors that calls into question the presumed role played by some of these hormones and reveals unexpected effects of others. These recent findings from the mutant mouse models are integrated and placed in context of the wider literature on endocrine-immune system interactions. The hypothesis that will be developed is that, with the exception of a role for thyroid hormones in B cell development, PRL, GH, and IGF-I are not obligate immunoregulators. Instead, they apparently act as anabolic and stress-modulating hormones in most cells, including those of the immune system.

NZB mice exhibit a primary T cell defect in fetal thymic organ culture.

Defects in T cell development have been suggested to be a factor in the development of systemic autoimmunity in NZB mice. However, the suggestion of a primary T cell defect has often been by extrapolation, and few direct observations of T cell precursors in NZB mice have been performed. Moreover, the capacity of NZB bone marrow T cell precursors to colonize the thymus and the ability of the NZB thymic microenvironment to support T lymphopoiesis have not been analyzed. To address this important issue, we employed the fetal thymic organ culture system to examine NZB T cell development. Our data demonstrated that NZB bone marrow cells were less efficient at colonizing fetal thymic lobes than those of control BALB/c or C57BL/6 mice. In addition, NZB bone marrow cells did not differentiate into mature T cells as efficiently as bone marrow cells from BALB/c or C57BL/6 mice. Further analysis revealed that this defect resulted from an intrinsic deficiency in the NZB Lin-Sca-1+c-kit+ bone marrow stem cell pool to differentiate into T cells in fetal thymic organ culture. Taken together, the data document heretofore unappreciated deficiencies in T cell development that may contribute to the development of the autoimmune phenotype in NZB mice.

Abnormal thymic expression of epithelial cell adhesion molecule (EP-CAM) in New Zealand Black (NZB) mice.

New Zealand Black (NZB) mice have been well documented to have a variety of thymic epithelial cell microenvironmental abnormalities, including disruption of corticoepithelial cell networks and medullary cell clusters. These abnormalities of the thymic stromal network are particularly important because similar observations have been noted in other models of murine lupus. Thymic epithelial cells, a key component of the microenvironment, play an important role in selection of the mature T cell receptor repertoire. Recently, a homotypic calcium-independent human and murine epithelial cell adhesion molecule, Ep-CAM, has been described which is located at the thymocyto-cortical cell junction. The function of Ep-CAM is still unclear but its unique location within the thymus suggests that it is critical in the process of providing maturation signals. Consequently, we examined the thymic expression of Ep-CAM in a series of autoimmune prone mice by thymic distribution of Ep-CAM in NZB, NZW, NZB/W, BXSB-Yaa, MRL- lpr/lpr, C3H- gld/gld and the control strains BALB/c, C57BL6, C3H and MRL(+/+), by immunohistology and flow cytometry. Interestingly, NZB mice are similar to control mice from day 4 to 2 weeks of age, having a very low expression of Ep-CAM at the thymocyto-cortical junction. In control strains, there is a marked increased in expression of Ep-CAM beginning at 5 weeks of age. In contrast, NZB mice fail to show significant expression of Ep-CAM even well into adulthood. This abnormality of NZB mice was also noted in NZB/W F1 and BXSB mice, but not MRL- lpr/lpr or C3H- gld/gld mice. Given the potential importance of Ep-CAM in thymic selection, this study provides important evidence that a defective stromal microenvironment is likely to be of etiological significance in the susceptibility of NZB to autoimmune disease.

Proliferation of bone marrow pro-B cells is dependent on stimulation by the pituitary/thyroid axis.

The frequency and absolute number of pro-B, pre-B, and B cells in the bone marrow of the hypothyroid strain of mice are significantly reduced compared with those of their normal littermates. To investigate why this is the case, various B cell developmental processes were examined in the thyroid hormone-deficient mice. These studies revealed that the frequency of pro-B cells in the S-G2/M phase of the cell cycle was significantly reduced in hypothyroid mice. That thyroid hormone deficiency was responsible for this proliferation defect was established by demonstrating that treatment of hypothyroid mice with thyroxine resulted in a specific increase in the frequency and total number of cycling pro-B cells. The latter effect was paralleled by increases in the frequency and number of bone marrow B lineage cells. Additional in vitro experiments revealed that at least some thyroid hormone effects were directly mediated on the bone marrow. Taken together, these data demonstrate that thyroid hormones are required for normal B cell production in the bone marrow through regulation of pro-B cell proliferation and establish a role for the pituitary/thyroid axis in B cell development.

Regulation of B and T cell development by anterior pituitary hormones.

Hormones produced by the anterior pituitary gland have been implicated in the regulation of primary lymphocyte development. In order to identify endocrine factors involved in that process, several strains of mice with genetic defects resulting in a selective impairment in the production of one or more anterior pituitary-derived hormones have been analysed. This study has resulted in the classification of endocrine hormones into the following four categories: (i) hormones such as prolactin with no apparent effects on primary lymphopoiesis; (ii) anabolic hormones such as growth hormone and insulin-like growth factor-I whose stimulatory effects on primary lymphopoiesis are non-lineage-specific and related to their actions as systemic mediators of growth and/or differentiation; (iii) hormones such as thyroid hormones that have an obligate role in primary B lymphopoiesis; and (iv) hormones such as oestrogens that act as negative regulators of lymphopoiesis.

Effects of insulin-like growth factor administration and bone marrow transplantation on thymopoiesis in aged mice.

There has been considerable interest in using hormone replacement therapy to rejuvenate the involuted thymus during aging. GH and insulin-like growth factor-I (IGF-I), a mediator of GH actions, have been of particular interest because of their thymopoietic effects and the fact that their serum concentrations decline during aging. However, treatment of aging rodents with either GH or IGF-I does not restore thymus cellularity to levels present in young animals, suggesting that additional defects might limit the magnitude of their effects. In particular, deficiencies have been reported to accumulate in the bone marrow T cell precursor compartment during aging. In view of this, 18-month-old mice were administered either recombinant IGF-I, bone marrow cells from young mice, or a combination of IGF-I and young bone marrow cells. Thymus cellularity in the latter group of mice was significantly higher than in animals treated with hormone or bone marrow transplantation alone, suggesting that optimal therapies for restoring thymus cellularity must address both endocrine and hematopoietic defects that accumulate during aging. Results from in vitro studies using fetal thymic organ cultures suggest that IGF-I acts by potentiating thymic colonization by bone marrow T cell precursors and/or that the hormone affects some other event soon after thymus colonization.

Primary B cell development is impaired in mice with defects of the pituitary/thyroid axis.

There has been considerable speculation that hormones produced in the anterior pituitary gland act as positive regulators of primary B cell development in the bone marrow. In order to identify endocrine factors that have such a role, B cell differentiation was examined in a panel of mice with genetic mutations that result in compromised production of one or more anterior pituitary hormones. This analysis demonstrated that the frequency of B lineage cells is significantly reduced in the dwarf and hypothyroid strains of mice, which have defects in the pituitary/thyroid axis, and that the production of normal numbers of pre-B cells is particularly dependent upon thyroid hormones. B cell development was normal in Little and IGF-I knockout animals, which have defects in the production of growth hormone and/or insulin-like growth factor I. The dependence of B lymphopoiesis on thyroid hormones appeared to be specific for that lineage, as myelopoiesis and thymopoiesis were normal in dwarf and hypothyroid mice. In addition to describing a specific endocrine hormone involved in the regulation of B cell development, these data provide evidence that normal production of bone marrow B lineage cells is dependent on extramedullary signals.

Myonuclear apoptosis in dystrophic mdx muscle occurs by perforin-mediated cytotoxicity.

Myonuclear apoptosis is an early event in the pathology of dystrophin-deficient muscular dystrophy in the mdx mouse. However, events that initiate apoptosis in muscular dystrophy are unknown, and whether elimination of apoptosis can ameliorate subsequent muscle wasting remains a major question. We have tested the hypothesis that cytotoxic T-lymphocytes initiate myonuclear apoptosis in dystrophic muscle, and examined whether perforin-mediated cytotoxicity plays a role in the pathophysiology of muscular dystrophy. Mdx mice showed muscle invasion by cytotoxic T cells and helper T cells at the onset of histologically detectable muscle fiber pathology. At this time, perforin-expressing cells were also present at elevated concentration. Mdx mice depleted of CD8(+) cells showed a significant reduction of apoptotic myonuclei concentration and a reduction in necrosis, judged by macrophage invasion of muscle fibers. Double-mutant mice, deficient in dystrophin and perforin, showed nearly complete absence of myonuclear apoptosis, and a significant reduction in the concentration of macrophages in the connective tissue surrounding muscle fibers. However, muscle fiber invasion by macrophages was not reduced significantly in double mutant mice. Thus, cytotoxic T-lymphocytes contribute significantly to apoptosis and necrosis in mdx dystrophy, and perforin-mediated killing is primarily responsible for myonuclear apoptosis.

Thymocyte development in vitro: implications for studies of ageing and thymic involution.

Functional defects that accumulate in the T cell compartment are thought to be responsible for the pronounced immunodeficiency that develops during ageing, and reduced production of T cells by the thymus as it undergoes involution has been suggested to contribute to this phenomenon. Understanding the mechanisms responsible for thymic involution requires a thorough knowledge of how thymopoiesis is regulated. Obtaining such information is dependent upon the availability of defined experimental systems that permit analysis of thymopoiesis at the cellular and molecular levels. Recent advances have been made in the development of such human and murine in vitro systems, and their analysis has the potential to identify thymic microenvironmental signals that regulate T cell production. This information should, in turn, provide a basis for understanding changes in thymopoiesis that occur during ageing. The features of these culture systems are reviewed in this article, and their potential application to the study of T cell production during ageing is discussed.

Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene.

Prolactin (PRL) has been implicated in numerous physiological and developmental processes. The mouse PRL gene was disrupted by homologous recombination. The mutation caused infertility in female mice, but did not prevent female mice from manifesting spontaneous maternal behaviors. PRL-deficient males were fertile and produced offspring with normal Mendelian gender and genotype ratios when they were mated with heterozygous females. Mammary glands of mutant female mice developed a normal ductal tree, but the ducts failed to develop lobular decorations, which is a characteristic of the normal virgin adult mammary gland. The potential effect of PRL gene disruption on antigen-independent primary hematopoiesis was assessed. The results of this analysis indicated that myelopoiesis and primary lymphopoiesis were unaltered in the mutant mice. Consistent with these observations in PRL mutant mice, PRL failed to correct the bone marrow B cell deficiency of Snell dwarf mice. These results argue that PRL does not play any indispensable role in primary lymphocyte development and homeostasis, or in myeloid differentiation. The PRL-/- mouse model provides a new research tool with which to resolve a variety of questions regarding the involvement of both endocrine and paracrine sources of PRL in reproduction, lactogenesis, tumorigenesis and immunoregulation.