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.

SummaryHyperbaric oxygen (HBO) treatment has been found to improve healing in living tissues, especially those poor in oxygen. The effects of HBO have also been tested in rat experiments. However, oxygen partial pressure in rat's arterial blood is normally about twice that in humans. Disregarding this, a human HBO protocol has been applied in previous rat experiments with HBO. Laser Doppler flowmetry (LDF) is a non-invasive means for measuring blood flow. Using LDF, we measured the blood perfusion rate in rats receiving HBO, according to a modified protocol, in a region of healing soft tissue with bone defect. The results indicate that, in rats, shorter HBO treatment with high O 2 pressure can significantly improve the blood flow of healing tissues. In this study, an elevated blood perfusion rate was still evident 2 weeks after the ending of HBO therapy, which indicates improved revascularization in the wound area. A short HBO protocol would save time and effort in future HBO experiments on rats.

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Mice homozygous for the scid (severe combined immunodeficiency) mutation are generally unable to produce B lymphocytes, a condition attributed to defective rearrangement of immunoglobulin genes in precursor B cells. Some early B-lineage cells are present in the bone marrow (BM), however. In scid mice, we defined three subsets of early progenitor B cells lacking mu heavy chains (pro-B cells) based on the expression of terminal deoxynucleotidyl transferase (TdT) and B220 glycoprotein: (a) early pro-B cells (TdT+B220-), (b) intermediate pro-B cells (TdT+B220+), and (c) late pro-B cells (TdT-B220+). Double immunofluorescence labeling of BM cell suspensions has shown normal numbers of early and intermediate pro-B cells, substantially reduced numbers of late pro-B cells, and an absence of pre-B cells and B cells. Early and intermediate pro-B cells accumulated in metaphase in near- normal numbers after intraperitoneal (IP) vincristine administration. B220+ pro-B cells have been localized in BM sections by the binding of intravenously (IV) administered 125I monoclonal antibody (MoAb) 14.8, detected by light and electron microscope radioautography. Many B220+ cells were located peripherally in the bone-lining cell layers associated with stromal reticular cells. More centrally located B220+ cells were frequently associated with macrophages containing prominent cytoplasmic inclusions. Occasional B220+ cells were present in venous sinusoids. These results demonstrate that many pro-B cells in scid mice occupy microenvironments in the BM near the surrounding bone. The pro-B cells maintain normal rates of production during stages of presumptive mu heavy-chain gene rearrangement, apparently unaffected by the absence of a mature B cell pool. Nearly all defective cells then abort at the late pro-B cell stage and are deleted, apparently by macrophages. The findings contribute to models of in vivo differentiation, regulation, localization, and selection of early B-lineage cells in the BM.

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A single injection of pristane was given i.p. to plasmacytoma-susceptible BALB/cAn mice. At intervals up to 6 mo thereafter, immunofluorescence labeling of intranuclear terminal deoxynucleotidyl transferase (TdT), cell surface B220 glycoprotein, cytoplasmic mu-chains of IgM (c mu), and surface mu-chains (s mu), together with mitotic arrest techniques, were used to quantitate the in vivo population dynamics of precursor B cells in the bone marrow. TdT-expressing pro-B cells (TdT+B220-, TdT+B220+), before the expression of mu-chains, showed sustained increases in both population size and the number of cells flowing through mitosis per unit time. In contrast, populations of pre-B cells (c mu + s mu -) and B cells (s mu +) were consistently depressed for long periods of time, including the phase of plasmacytoma formation. Precursor B cells in DBA/2 mice, a plasmacytoma-resistant strain, showed similar responses to pristane treatment. The results demonstrate that a single injection of pristane, which greatly increases the demand for macrophage activity in the peritoneal space, causes sustained distant alterations in B cell lymphopoiesis in the bone marrow; specifically, a prolonged increased proliferation of pro-B cells coupled with a depression and a exaggerated loss of pre-B cells and B cells. The protracted stress on B cell lymphopoiesis may be a predisposing factor in the subsequent development of c-myc-activating chromosomal rearrangements that play a critical role in plasmacytomagenesis.

To examine the in vivo role of c-kit receptor in B lymphopoiesis we have evaluated precursor B cell populations expressing c-kit in mouse bone marrow and the effects on B cell genesis of administering a neutralizing anti-c-kit mAb, ACK2. Double immunofluorescence labeling and mitotic arrest were used to examine bone marrow cells from BALB/c mice. Almost one-half of TdT+ cells and one-quarter of B220+ cells coexpressed c-kit, mainly at low intensities, and were actively proliferating in vivo. c-kit+ cells that lacked B lineage markers expressed c-kit in high intensities and entered mitosis at an exceptionally rapid rate. In ACK2-treated mice, erythroid and granulocytic cells were almost completely absent from the bone marrow, whereas, in contrast, B lymphopoiesis was stimulated. Pre-B cells expressing cytoplasmic mu-chains; as well as TdT+B220+ cells before mu expression, were increased two- to fourfold in number and production rate. IgM-bearing B lymphocytes were increased in bone marrow and spleen. The results demonstrate that many early precursor B cells in mouse bone marrow constitutively express c-kit receptor. The failure of ACK2 treatment to block B lymphopoiesis, however, suggests that c-kit receptor function is not essential for precursor B cell development in vivo, but can be replaced by alternative signaling systems. The stimulation of B cell genesis by ACK2 treatment may reflect a conferred advantage in the competition for microenvironmental factors which underlies the balance between B lymphopoiesis and other hemopoietic lineages in vivo.