Dennis G. Osmond

Population Dynamics of Bone Marrow B Lymphocytes

The dynamic concepts of lymphocyte populations which heralded the era of modern cellular immunobiology have been generally substantiated by recent studies and are still being correlated with functional properties. B lineage cells in the bone marrow are dynamically heterogeneous: A large majority are newly-formed, rapidly renewed cells, continuously produced from precursor cells within the bone marrow and disseminated during a terminal maturation phase via the blood stream. These cells develop low densities of sIgM in the extravascular bone marrow parenchyma and may undergo some further maturation within bone marrow sinusoids. The rate of production of bone marrow B cells appears to depend partly on the total load of exogenous agents to which the individual is exposed. Bone marrow lymphocyte production maintains a population of rapidly renewed virgin B cells in the peripheral lymphoid tissues. A small proportion of these cells apparently may be selected to enter a long-lived pool of B cells if suitably activated. By continuously creating novel clonotypes this process potentially can anticipate new antigen challenges and allow the immune system to build up a repertoire of antigen specificities most appropriate to the individuals changing environment throughout life. A minority of B lymphocytes in the bone marrow comprises slowly renewed, long-lived cells which enter and leave the bone marrow parenchyma as a selective part of the recirculating lymphocyte pool in the blood stream. Their role in the bone marrow is unknown. They include antigen-specific B memory cells, yet these are not activated within the bone marrow itself. No regulatory role has yet been directly demonstrated. Recently activated B cells enter from the spleen after secondary antigenic stimulation to develop into antibody-producing cells within the bone marrow. In assessing the significance of any phenotypically or functionally distinct B cell subset in the bone marrow, a basic consideration is to assign the subset to one of the foregoing dynamic categories. Within a given category cells may represent one stage in a time sequence of development. The bone marrow also produces lymphocytes of as yet uncertain lineage and contains selected subsets of T cells. The roles of these cells in cytotoxic, regulatory, or other events remain to be elucidated.

Murine B lymphopoiesis: towards a unified model

Abstract Despite much progress in elucidating the cellular stages of B-cell development, the use of diverse phenotypic criteria and terminologies has hampered cross-reference between various models of B-cell differentiation. Here, Dennis Osmond, Antonius Rolink and Fritz Melchers combine their findings to provide a common phenotypic model of B-cell development in mouse bone marrow.

Proliferation kinetics and the lifespan of B cells in central and peripheral lymphoid organs

Abstract Comprehensive models of the production and loss encountered during the differentiation of precursor B cells, and the use of new methods to re-examine the heterogeneity of B-cell lifespans have resulted in recent progress in the genesis and regulation of the B-cell system in vivo.

Apoptosis and Macrophage-Mediated Cell Deletion in the Regulation of B Lymphopoiesis in Mouse Bone Marrow

Studies of cell population dynamics and microenvironmental organization of B lymphopoiesis in the bone marrow of normal mice and in various genetically modified states have shown that cell loss, involving processes of apoptosis and macrophage-mediated cell deletion, is a prominent feature of the primary genesis of B lymphocytes. Balanced against the influence of proliferative stimulants, the programmed death of precursor B cells provides a quantitative control, determining the magnitude of the final output of functional B lymphocytes to the peripheral immune system. The cell loss mechanisms can be readily set in motion by external or systemic influences, making the B-cell output particularly vulnerable to suppression by ionizing irradiation, stress or other systemic mediators. In addition, however, cell loss exerts an important quality control in the formation of the primary B-cell repertoire. The combination of apoptosis and macrophage-mediated deletion, acting at successive stages of B-cell differentiation, efficiently eliminates many precursors having non-productive Ig gene rearrangements, cell cycle dysregulations, and certain autoreactive Ig specificities. Outstanding areas of further work abound. Important questions concern the nature of mechanisms which underlie the processes of B-cell apoptosis and macrophage deletion in bone marrow, the microenvironmental signals involved in B-cell life or death decisions and genetic factors which may override these B-cell culling mechanisms. The answers will be relevant to problems of autoimmune disease, humoral immunodeficiency and B-cell neoplasia.

Apoptosis and its modulation during B lymphopoiesis in mouse bone marrow

Summary: Studies in normal, gene‐deleted, transgenic and mutant mice have examined apoptotic cell death and its role in B lymphopoiesis in bone marrow. Apoptotic activity has been quantitated among phenotypically defined populations of precursor B cells using flow cytometry of apoptotic cells and an established model of B‐cell development. In normal mice, the frequencies of apoptotic cells (apoptotic index) and accumulation of apoptotic cells during short‐term culture (apoptotic rate) are maximal at around the pro/pre‐B‐cell transition and among immature B lymphocytes. The brief period between onset of apoptosis and clearance by macrophages (apoptotic transit time) is similar for most precursor B‐cells. Apoptosis‐modulating factors produce substantial changes in apoptotic activity among pro‐B and pre‐B cells, associated with altered expression of bcl‐2 family proteins. Pro‐B‐cell apoptosis, normally extensive, is markedly suppressed in the absence of p53. Complete pro‐B‐cell abortion in RAG‐2 deletion provides an assay for apoptotic fractions in other experimental systems. Pre‐B‐cell apoptosis is enhanced by deficiencies of interleukin (IL)‐7, Abl protooncogene or colony‐stimulating factor (CSF)‐1 and overexpression of heat‐stable antigen, and is inhibited by IL‐7 and p190bcr/abl transgenes. CSF‐1 and melatonin administration inhibit pre‐B‐cell apoptosis, probably via stromal cell stimulation. Such apoptotic modulation has implications for B‐cell homeostasis, quality control, immunodeficiency and neoplasia.

Melatonin inhibits apoptosis during early B-cell development in mouse bone marrow.

The pineal secretory product, melatonin, exerts a variety of effects on the immune system. Administration of melatonin stimulates cell‐mediated immunity, particularly by inhibiting apoptosis among T lymphocytes in the thymus and inducing production of T‐cell‐derived cytokines. However, its possible effects on the humoral immune system are unclear. In the present study, we have examined whether melatonin may influence the in vivo development of B lymphocytes in mouse bone marrow, a process in which apoptosis is normally a prominent feature. Double immunofluorescence labeling and flow cytometry were used to quantitate phenotypically defined precursor B‐cell and mature B‐cell populations and their apoptotic rates in bone marrow of mice fed either melatonin‐containing or control diet for 16 days from 9 wk of age. In short‐term bone marrow cultures, the incidence of apoptosis among large pre‐B cells, including cells expressing the Λ5 component of pre‐B‐cell receptor, was markedly reduced in melatonin‐treated mice, associated with an increase in the absolute number of large pre‐B cells in bone marrow. In contrast, apoptosis of earlier precursor B cells and mature B lymphocytes did not differ from control values. The results indicate that orally administered melatonin can substantially promote the survival of precursor B cells in mouse bone marrow. Melatonin treatment may thus boost the survival of newly formed B cells mediating humoral immunity.

Regulation of bone marrow lymphocyte production: III. Increased production of B and non-B lymphocytes after administering systemic antigens

To examine the influence of exogenous stimuli on the genesis of lymphocytes in mouse bone marrow, the production rate and subsets of marrow lymphocytes were examined after a systemic injection of sheep red blood cells (SRBC). Radioautographic analysis after either pulse labeling or infusion of [3H]thymidine revealed a pronounced increase in the number of newly formed small lymphocytes appearing in the marrow, maximal 4-5 days after SRBC injection and dose related. The resulting expansion of the marrow lymphocyte population included both immature B cells and null cells, as shown by cell surface and cytoplasmic markers. Similar stimulation of marrow lymphocyte production followed an injection of either bovine serum albumin or mineral oil. No comparable stimulation occurred in either the thymus or the spleen. The results demonstrate that antigens and nonspecific irritants can exert a central effect in the bone marrow, producing a surge in the production of both primary B and non-B lymphocytes. The possible role of external stimulants in determining the normal rate of bone marrow lymphocyte production is discussed.

Cell Cycle Quiescence of Early Lymphoid Progenitors in Adult Bone Marrow

Lymphocyte production in bone marrow (BM) requires substantial cell division, but the relationship between largely quiescent stem cells and dividing lymphoid progenitors is poorly understood. Therefore, the proliferation and cell cycle status of murine hematopoietic progenitors that have just initiated the lymphoid differentiation program represented the focus of this study. Continuous bromo‐2′‐deoxyuridine (BrdU) incorporation and DNA/RNA analysis by flow cytometry revealed that a surprisingly large fraction of RAG‐1+c‐kithi early lymphoid progenitors (ELPs) and RAG‐1+c‐kitlo pro‐lymphocytes (Pro‐Ls) in adult BM were in cell cycle quiescence. In contrast, their counterparts in 14‐day fetal liver actively proliferated. Indeed, the growth fraction (cells in G1‐S‐G2‐M phases) of fetal ELPs was on average 80% versus only 30% for adult ELPs. After 5‐fluorouracil treatment, as many as 60% of the adult ELP‐enriched population was in G1‐S‐G2‐M and 34% incorporated BrdU in 6 hours. Transcripts for Bcl‐2, p21Cip1/Waf1, and p27 Kip1 cell cycle regulatory genes correlated inversely well with proliferative activity. Interestingly, adult lymphoid progenitors in rebound had the high potential for B lymphopoiesis in culture typical of their fetal counterparts. Thus, lymphocyte production is sustained during adult life by quiescent primitive progenitors that divide intermittently. Some, but not all, aspects of the fetal differentiation program are reacquired after chemotherapy.

Two models of murine B lymphopoiesis: a correlation

During B cell genesis in mouse bone marrow (BM), precursor B cells pass through a series of developmental stages that have been defined by changes in expression of various marker molecules. The use of dissimilar phenotypic criteria in different laboratories, however, has led to the formulation of disparate models of B lymphopoiesis not fully reconciled with one another. We have directly compared two such models, one based on expression of intracellular μ heavy chain of IgM (cμ) and terminal deoxynucleotidyl transferase (TdT), the other monitoring cell surface leukosialin (CD43), heat‐stable antigen (HSA; CD24) and the ectopeptidase BP‐1. Each model uses cell surface B220 glycoprotein (CD45RA) to denote the B cell lineage. We have examined the cellular composition of four sorted BM fractions by immunofluorescent labeling of CD43, HSA and BP‐1, using immunofluorescence microscopy of cytocentrifuged fractions to quantitate precursor B cell populations expressing either cμ or TdT. The results reveal a range of B cell differentiation stages within individual sorted BM fractions, providing a cross‐reference between these two analytical methods and contributing to a unified model of B cell development in mouse BM.

The localization of B lymphocytes in mouse bone marrow: Radioautographic studies after in vivo perfusion of radiolabelled anti-IgM antibody

An in vivo cell surface labelling technique using radioautography has been developed to visualise the distribution of IgM-bearing B lymphocytes within the bone marrow. Anaesthetized 3-week-old mice were perfused via the common iliac artery with: (1) serum-containing medium (SCM), (2) 125I-labelled anti-IgM antibody in SCM, (3) SCM, and (4) fixative. In radioautographic sections of femoral marrow labelled surface IgM+ cells were observed either singly or in small clusters throughout the extravascular haemopoietic marrow cords. Binding specificity was demonstrated by the displacement of 125I-anti-IgM labelling by excess anti-IgM and by the binding of perfused 125I-anti-H-2Kk antibody in CBA/J (H-2Kk) mice but not in C57BL/6 (H-2Kb) mice. Quantitative analysis of radioautographic sections revealed an even distribution of labelled cells throughout CBA/J marrow perfused with 125I-anti-H-2Kk, indicating a uniform accessibility of perfused antibody to cells in the haemopoietic cords. This labelling pattern contrasted with that in 125I-anti-IgM perfused animals in which surface IgM+ cells, although widely distributed in the bone marrow, showed areas of concentration, speculatively clones of maturing B lymphocytes. This method of labelling surface IgM and other cell markers in situ provides an approach to study the microenvironment of B lymphocyte genesis in the bone marrow.