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.

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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 individual's 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.

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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.

B lymphocyte precursor cells expressing B220 glycoprotein have been examined in mouse bone marrow (BM) by the in vivo binding of monoclonal antibody (mAb) 14.8 visualized by light and electron microscope radio autography. Young mice were injected intravenously with 125I-labeled mAb 14.8 and then perfused to remove unbound antibody. Quantitative analysis of radioauto graphic sections of femoral BM revealed many labeled mAb 14.8-binding cells which were situated both singly and in groups throughout the extravascular BM parenchyma. Groups of large 14.8+ cells were located in patchy areas in the peripheral regions of the BM near the endosteum. These cells were shown to include proliferating precursor B cells by using mice given vincristine sulfate to stop cells in metaphase and mice treated from birth with anti-IgM antibodies to delete mature B lymphocytes. Electron microscopy revealed clusters of 14.8+ cells intimately associated with the processes of stromal reticular cells. Other 14.8+ cells were in close contact with macrophages; in some instances the intervening cell membranes were indistinct and the macrophages contained 14.8+ material in their cytoplasm. In addition, 14.8+ small lymphocytes were highly concentrated within the lumen of some sinusoids. The present method of detecting B lineage precursor cells in situ has led to a working model of the microenvironmental organization of primary B cell genesis in vivo. The model proposes (a) a centrally directed sequence of differentiation initiated by early precursor cells situated peripherally near the surrounding bone; (b) close associations between precursor B cells and stromal reticular cells; (c) deletion of ineffective B cells by macrophages, and (d) an intravascular maturation phase before B lymphocytes are finally delivered into the blood stream.

Thymidine-H3 was injected into the femoral and tibial marrow (labeled marrow) of guinea pigs while the hind limb circulation was arrested temporarily and non-radioactive thymidine was administered systemically. Blood and lymphoid tissue radioautographs were subsequently examined for the presence of marrow-derived labeled cells.Small lymphocytes in the labeled marrow showed a wave of labeling, maximal a t two to three days. Concurrently, labeled small lymphocytes appeared i n the blood and lymphoid tissues, mainly the spleen and mesenteric lymph node. Their numbers were greatest at four to five days, and declined rapidly thereafter. At first they appeared predominantly i n the splenic red pulp and throughout the lymph node cortex, including the subcapsular sinus. By four to five days they were also concentrated in the splenic white pulp, including periarteriolar lymphoid sheaths, and in the lymph node medullary cords. They were detected within medullary sinuses, hilar lymphatics and thoracic duct lymph. Labeled monocytes and large lymphoid cells also appeared i n the blood and lymphoid tissues, mainly in the spleen.It is concluded that bone marrow is a major source of circulating newly-formed small lymphocytes many of which migrate rapidly into the spleen and mesenteric lymph node.It is now well established that considerable numbers of small lymphocytes are produced continuously in the bone marrow of guinea pigs (Osmond and Everett, '64; Harris and Kugler, '65; Osmond, '67) and rats (Everett and Caffrey, '67). Radioautographic studies, utilising DNA-labeling with thymidine-H3, have demonstrated that the large population of marrow small lymphocytes (Hudson, Osmond and Roylance, '63; Yoffey, '66) is composed entirely of newly-formed cells (Everett, Caffrey and Rieke, '64; Craddock, '65; Everett and Tyler (Caffrey), '67) which are renewed rapidly by the proliferation of precursor cells within the marrow (Osmond and Everett, '64; Harris and Kugler, '65; Osmond, '67; Everett and Caffrey, '67; Yoshida and Osmond, '69). Some small lymphocytes have been shown to enter the blood stream from the marrow in both guinea pigs (Osmond, '65 and '66; Hudson and Yoffey, '66; Linna and Liden, '69) and rats (Everett and Caffrey, '67) but little has been learned of their subsequent life history and fate. In determining the functional r61e of marrow small lymphocytes in vivo an important question therefore concerns their precise pathways of migra-ANAT. REC., tion and their topographical interrelationships with other lymphocyte populations.In the present experiments the release of newly-formed small lymphocytes from the marrow and their localisation within other lymphoid organs has been examined radioautographically after selective marrow labeling with thymidine-H3. Using a technique developed previously in the guinea pig (Osmond, '65, '66), labeling was confined at first to the DNA-synthesising cells of tibial and femoral marrow by injecting thymidine-H3 directly into the medullary cavities while the local circulation was ...