Regulated progression of B lymphocyte differentiation from cultured fetal liver.

Lymphoid fetal liver cultures (LFLC) are long-term, nontransformed cultures of early B lymphoid lineage cells which appear developmentally blocked at the pre-B stage in vitro. When injected into severe combined immunodeficient (SCID) mice, cells from LFLC could reconstitute splenic B lymphocytes and serum IgM. T lymphocyte reconstitution was not observed and serum IgG levels were very low. IgG3 was the predominant gamma subisotype in the serum of the LFLC-reconstituted mice, indicating impaired class switching in these B lymphocytes. When thymocytes were coinjected with LFLC, the B lymphocytes were able to class switch fully and respond to T-dependent antigens. These serological responses were heterogeneous. This experimental system allows separation of three B lymphocyte developmental stages: early differentiation in vitro, progression to IgM secretion in vivo, and late differentiation dependent upon mature T lymphocytes in vivo. The unique advantage of this system is the ability to regulate the B lymphocyte developmental pathway in a defined, stepwise manner.

Generation of biliary lesions after transfer of human lymphocytes into severe combined immunodeficient (SCID) mice.

Human PBL have been reported to reconstitute B and T cells as well as human serum Ig in mice with severe combined immunodeficiency disease (SCID). To confirm these observations and attempt the transfer of an autoimmune disease to the immunodeficient animals, groups of SCID mice received an injection of PBL from patients with primary biliary cirrhosis (PBC) or from normal volunteers. By 8 wk after the injection of 10-42 x 10(6) PBL into the mice, human lymphoid cells were detected in the spleen of approximately half of the animals and all had detectable serum levels of human IgG. Moreover, the sera of SCID mice that received cells from patients with PBC contained human antimitochondrial antibodies (AMA) to dihydrolipoamide acetyltransferase, the major mitochondrial autoantigen of PBC. Histologically, a human mononuclear cell infiltrate was present around the portal areas of the liver and inflammation, bile duct atypica, and necrosis of bile duct cells were observed. While the biliary lesions in the SCID recipients of PBC cells were more severe, a mononuclear infiltrate was clearly evident in mice that received cells from normal donors, suggesting the presence of a graft-vs.-host-like disease. While these data are the first to describe an animal model with both the humoral and cellular characteristics of PBC, they also raise an interesting question regarding the preferential localization of lymphoid cells to the biliary system.

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.

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.

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.

Pan/E2A expression precedes immunoglobulin heavy-chain expression during B lymphopoiesis in nontransformed cells, and Pan/E2A proteins are not detected in myeloid cells.

A newly developed rat long-term bone marrow culture system was used to study the role of Pan/E2A basic helix-loop-helix transcription factors during B-cell development. In this system, B-lymphocyte progenitors actively differentiate into mature B cells. Monoclonal (Yae) and polyclonal (anti-Pan) antibodies were employed to characterize the expression of Pan proteins by Western blot assay during hematopoiesis and to examine the components of immunoglobulin heavy-chain gene enhancer element-binding species by electrophoretic mobility shift assay. During B-cell development, the appearance of Pan/E2A proteins preceded the expression of immunoglobulin heavy-chain protein. A Pan-containing immunoglobulin heavy-chain enhancer element (mu E5)-binding species (BCF1), composed of immunoreactive Pan-1/E47 but not Pan-2/E12, was observed concomitantly with the detection of Pan/E2A proteins. In addition to BCF1, other mu E5-binding species were detected which were not recognized by the Yae antibody. Two of these species were present in primary B-lymphocyte and myeloid cultures and were recognized by an anti-upstream stimulatory factor antiserum. Although Pan/E2A proteins have been proposed to be ubiquitous, Pan/E2A proteins were not detected in primary myeloid cultures composed mainly of granulocytes and macrophages or in the macrophage cell line J774. The absence of Pan/E2A proteins in differentiated myeloid cells correlated with low steady-state levels of Pan/E2A RNA. However, Pan/E2A proteins were present in a promyeloid cell line, 32DCL3, suggesting that extinction of Pan/E2A expression may play a role in myelopoiesis.

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.

1,25-Dihydroxyvitamin D3 inhibits myelopoiesis but not B-lymphopoiesis in long-term bone marrow cultures.

The biologically active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) has been demonstrated to have differentiative and antiproliferative effects on myeloid tumors of human or murine origin. Its effects on normal murine hemopoiesis were tested by addition of the seco-steroid to long-term bone marrow cultures optimized for either myelopoiesis or B-lymphopoiesis. The addition of 10(-8) M 1,25(OH)2D3, but not 10(-8) M 25(OH)D3, to myeloid bone marrow cultures (MBMC) resulted in a complete cessation of hemopoiesis by 4 weeks, because no hemopoietic cells or colony-forming units were detected. This result was observed whether or not the cultures were initiated and maintained in hydrocortisone. A potential effect of 1,25(OH)2D3 on the production of myeloid growth factors by adherent layer cells in the cultures was examined, but this function was not affected by 1,25(OH)2D3 treatment. Further, adherent layers that had been treated with 1,25(OH)2D3 for 3 weeks were capable of supporting myelopoiesis upon seeding with a stromal cell-depleted population of bone marrow cells. Transfer of MBMC to lymphoid bone marrow culture (LBMC) conditions results in the cessation of myelopoiesis and the initiation of B-cell production. Lymphopoiesis did not initiate in 1,25(OH)2D3-pretreated MBMC that were transferred to LBMC conditions, indicating that the pool of B-cell precursors present in MBMC had been depleted by exposure of MBMC to 1,25(OH)2D3. When 1,25(OH)2D3 (10(-8) M) was added to MBMC at the time of transfer to LBMC conditions, the seco-steroid did not affect induction of B-lymphopoiesis, although the overall cellularity was less in 1,25(OH)2D3-treated cultures than in control cultures.

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.

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.

Human, rat or mouse hybridomas secrete high levels of monoclonal antibodies following transplantation into mice with severe combined immunodeficiency disease (SCID).

Mice with severe combined immunodeficiency disease (SCID) have been investigated for their ability to grow xenogenic hybridomas of mouse, rat and human origin. Two rat X mouse hybridoma lines (187.1.10 and 3B9) and 1 mouse X mouse hybridoma (2D9) grown in pristane-treated SCID mice as ascites tumors showed a 100-200-fold increase in monoclonal antibody levels over the amount produced in vitro with a total yield up to 0.5 g of antibody per animal. A human X human hybridoma, CLL-11-D1, exhibited a 1000-fold increase in human immunoglobulin levels in ascites (1.3 mg/ml) as compared to that obtained in tissue culture. Analyses of the antibody protein in the SCID ascites produced by these hybridomas using protein electrophoresis, SDS polyacrylamide gel electrophoresis and high resolution isoelectric focusing indicated the antibodies were monoclonal and free from any contaminating immunoglobulins. Yields of monoclonal antibodies of over 90% purity could be obtained from the ascites by a single ammonium sulfate precipitation step. This study indicates that SCID mice provide several significant advantages over other in vivo methods for the production of pure monoclonal antibodies of human, rat, or mouse origin.

A colony assay system that detects B cell progenitors in fresh and cultured bone marrow.

This report describes a colony assay system, based on methods used to grow myeloid precursors in semisolid medium, in which B cell progenitors can be grown. The formation of these B cell progenitor colonies is dependent upon soluble mediators from a stromal cell line known to support B lymphopoiesis. In initial experiments a double layer culture system was employed in which target cells in methylcellulose medium were separated from an adherent layer of S17 stromal cells by an agar interface. Target cells were harvested from Dexter type long-term bone marrow cultures at a time after transfer to the lymphoid Whitlock-Witte conditions, when myeloid progenitors were depleted and mature B cells had not yet appeared. On day 15 of culture a colony could be identified that contained several hundred tightly clustered lymphoid cells. There was a linear relationship between the number of cells plated and the number of colonies that developed. Identically appearing colonies were also observed in agar using fresh bone marrow cells as targets with either an underlayer of S17 cells or S17 conditioned medium to potentiate colony growth. Lymphoid colonies derived from fresh bone marrow appeared on days 6 and 14 of growth. A proportion of the cells from the fresh or cultured marrow derived colonies expressed the B220 antigen and cytoplasmic mu heavy chains, but surface IgM was never observed. Cell depletion experiments on antibody coated plates demonstrated the colony forming unit to be B220 antigen positive, surface IgM negative, and replating experiments indicated the colonies were lymphoid restricted in their differentiative potential.

Generation of purified stromal cell cultures that support lymphoid and myeloid precursors.

Treatment of Dexter-type long-term bone marrow cultures with the antibiotic mycophenolic acid (MPA) eliminates all hemopoietic cells from the cultures, while a morphologically intact, adherent stromal cell layer is retained. The ability of these MPA treated stromal cell cultures to support long-term hemopoiesis was tested by seeding them with fresh bone marrow cells that had been passed through nylon wool. This procedure yields a relatively stromal cell depleted population of hemopoietic cells. An aliquot of 5 X 10(5) or 2.5 X 10(5) nylon wool passed bone marrow cells bearing the T6 chromosomal marker was seeded onto replicate MPA-treated stromal cell layers. The stromal cells stimulated the proliferation of the bone marrow cells, and nonadherent cells were present for up to 8 weeks of culture. Progenitors of granulocytes and macrophages (CFU-GM) were also present for this period of time despite weekly demi-depopulation, during culture feeding. Karyotypic analysis confirmed that the CFU-GM were derived from the reseeded population. Nylon wool-passed bone marrow cells seeded alone into empty flasks under identical conditions did not survive past 1 week. Cells from the reseeded cultures were also tested for early myeloid precursors (CFU-S) and injected into immunodeficient CBA/N mice to test for the presence of primitive B cell precursors. CFU-S were present in mice killed 11 days following injection of cells, and high levels of B cell colony-forming units (CFU-B) were present in mice 4 weeks post reconstitution. Further studies demonstrated that factors present in medium conditioned by the stromal cells could support the growth of CFU-GM. These data indicate that treatment of long-term bone marrow cultures with MPA results in a population of functional stromal cells.

Hemopoietic cell dysfunction in murine lupus.

Irregularities have appeared through almost the entire detectable range of hemopoiesis, from stem cells to functional mature populations in several models of murine lupus. The documentation of widespread abnormalities in many cell lineages implies the existence of a common, defective ancestor, perhaps the pluripotential hemopoietic stem cell. Of major concern are the microenvironmental pressures that may be driving hemopoiesis to its pathological state. The studies to date have not isolated the hemopoietic components from their cellular surroundings. Hence, the existence of a primary defect in any particular cell compartment is as yet an unanswerable point. Additionally, maternal forces must be considered as environmental factors whose consequences may extend into postnatal life. Also, the possible existence of hemopoietic cell influences on their environs are always present, creating a cellular ecosystem. As new techniques become available for the analysis of these cells, such as long-term cell culturing, more complete pictures of these murine models of autoimmunity will emerge.

Regulatory cells and cytokines involved in primary B lymphocyte production.

Based upon the above data, it is now possible to formulate a working model that defines the stages of B cell development on which stromal cells and their products act. During the initial stages of this process, pro-B cells which do not express Ig heavy or light chain protein or other non-Ig B lineage associated molecules develop into B220 and c mu expressing pre-B cells in response to a low (< 10 kD) molecular weight stromal cell derived factor. No defined interleukin or colony stimulating factor, including molecules such as KL and IL-7, can replace stromal cell conditioned medium in mediating this developmental step. There appears to be little cell proliferation associated with the differentiation of pro-B cells into pre-B cells. However, our data indicate that as precursors develop into B220 expressing B cell progenitors, they become sensitive to the proliferation stimulating effects of IL-7 and KL. These results are in accord with findings that progenitor cells that have undergone DJH rearrangements are particularly sensitive to KL and IL-7(18,19). The analysis of pre-B cells present in individual lymphoid colonies indicates that once cells have rearranged and expressed their Ig heavy chain genes, they are no longer sensitive to KL and IL-7. These observations are based on the fact that receptors for these cytokines are not expressed in stromal cell dependent pre-B cells and are consistent with kinetic studies showing that the maturation of pre-B cells into surface Ig expressing B lymphocytes is not dependent upon cell proliferation21.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of bone marrow stromal cell-surface antigens on myeloid and lymphoid cells.

Two monoclonal antibodies (MAb) that recognize cell-surface determinants present on bone marrow stromal cells have been generated. The 5B3 antibody recognizes a major histocompatibility class I-like molecule. This antigen is coexpressed on all bone marrow B-lineage cells and myeloid progenitors responsive to recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). The 9D3 antibody recognizes a protein similar in size to those encoded by the Ly-6 locus. A comparable bone marrow staining pattern and inhibition of 9D3 binding to bone marrow cells by anti-Ly-6C antibodies strongly suggests that the 9D3 antigen is Ly-6C. These data provide a further phenotypic characterization of bone marrow stroma and indicate that these cells express cell-surface determinants also present on myeloid and lymphoid cells.