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D G Osmond

Publications and source records attributed to D G Osmond.

At least 19 recordsLinked to original sources
PubMed1992

Highly restricted expression of a stromal cell determinant in mouse bone marrow in vivo.

B lymphocyte precursor cells in mouse bone marrow develop in close association with stromal cells which provide essential growth signals. To identify molecules that may normally play a role in this interaction we have examined the in vivo binding of a new monoclonal antibody (mAb) (KMI6) that recognizes a determinant on a bone marrow stromal cell line (BMS2) in vitro. Flow cytometric and radioautographic evaluations revealed that the antigen recognized by KMI6 is represented on the surface of an extremely small number of cells in bone marrow cell suspensions from adult mice. An apparent molecular mass of 110 kD was obtained by surface labeling of a stromal cell clone and immunoprecipitation. Purified mAb KMI6 labeled with 125I was then given intravenously to young C3H/HeJ mice. Unbound mAb was washed out by cardiac perfusion and femoral bone marrow was examined by light and electron microscope radioautography. KMI6 labeling was heavy on the plasma membrane of many stromal cells, especially those located towards the outer subosteal region. The KMI6-labeled stromal cells were usually associated with cells of lymphoid morphology which they often completely surrounded. The labeling was restricted to areas of stromal cell plasma membranes in contact with lymphoid cells. The lymphoid cells themselves, as well as macrophages and other hemopoietic cells, failed to bind mAb KMI6 significantly. Stromal cells in bone marrow depleted of hemopoietic cells by gamma-irradiation (9,5 Gy) bound mAb KMI6 at reduced intensity. The results demonstrate that the KMI6 determinant, a 110-kD protein, is expressed on bone marrow stromal cells in vivo. Its restriction to areas of interaction with lymphoid cells suggests a role in forming microenvironmental niches of B lymphopoiesis. The surface membrane of individual stromal cells may thus be functionally polarized towards interacting B cell precursors and other hemopoietic cells.

Animals↗
PubMed1992

Dynamics and localization of early B-lymphocyte precursor cells (pro-B cells) in the bone marrow of scid mice.

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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PubMed1992

Changes in the populations of null, NK1.1+, and Thy1lo lymphocytes in the bone marrow of tumor-bearing mice: effect of indomethacin treatment.

Mouse bone marrow produces many "null" lymphocytes which lack B and T lineage markers (B220-Thy1-). A subset of these cells expresses the natural killer (NK) cell marker, NK1.1. In addition, some rapidly renewed bone marrow lymphocytes express low intensities of Thy1 (Thy1lo). In view of their possible implication in tumor-host interactions these various cell populations have now been examined in mice injected with either the nonmetastatic Ehrlich ascites (EA) tumor or the Lewis lung carcinoma (LLc), a highly metastatic solid tumor. In each case, the number of null lymphocytes, as defined by a lack of radioautographic labeling of either B220 glycoprotein or Thy1, increased markedly in both the bone marrow and spleen. Treatment with the prostaglandin inhibitor, indomethacin, enhanced the increase in null cells in the bone marrow and spleen of LLc-bearing mice. The number of null small lymphocytes expressing NK1.1, as detected by combined radioautographic and immunoperoxidase techniques, increased almost 30-fold in LLc-bearing mice. The number of Thy1lo small lymphocytes increased in parallel with null cells during EA tumor growth. The findings accord with the hypothesis that the null lymphocyte population produced in mouse bone marrow includes newly formed NK lineage cells which sequentially express NK1.1 and Thy1lo. The present work demonstrates that the populations of null, NK1.1+, and Thy1lo lymphocytes in mouse bone marrow expand rapidly during the early growth of transplanted tumors, the initial increase in null lymphocytes apparently being curtailed by prostaglandin production. The results suggest that the production of null lymphocytes in mouse bone marrow is responsive to tumor development, possibly providing cells to be involved in tumor-host interactions.

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PubMed1991

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.

Age Factors↗
PubMed1991

Regulation of early precursor B cell proliferation in mouse bone marrow: stimulation by exogenous agents mediated by macrophages in the spleen.

An increase in pre-B cell proliferation and B lymphocyte production in mouse bone marrow has previously been shown to follow the administration of various foreign agents in vivo. The responses of early precursor B cells before the expression of mu chains (pro-B cells) have now been examined, using double immunofluorescence labeling for terminal deoxynucleotidyl transferase (TdT) and B220 glycoprotein as detected by monoclonal antibody 14.8. A single injection of sheep red blood cells (SRBC) was followed by an increase in the number of cells in three defined populations of early precursor B cells lacking mu chains (TdT+ 14.8- cells, TdT+ 14.8+ cells, and 14.8+ mu- cells) as well as cytoplasmic mu-bearing pre-B cells and surface mu-bearing B lymphocytes. An accompanying increase in proliferative activity was indicated by the numbers of 14.8+ mu- cells and pre-B cells which accumulated in metaphase after inducing mitotic arrest with vincristine. These effects were all abrogated either by treating mice with silica to depress macrophage function or by splenectomy. In mice given multiple injections of SRBC for 4 weeks the elevated levels of early precursor B cell production and B cell genesis were sustained. The work demonstrates that the in vivo production of early precursor B cells, putatively including those at the stage of Ig heavy chain gene rearrangement, can be stimulated by exposure to external agents acting indirectly by a silica-sensitive, spleen-dependent mechanism. The findings suggest that the level of pro-B cell proliferation and primary B cell genesis normally taking place in mouse bone marrow may reflect the level of exposure to potential stimulants in the external environment mediated by activation of splenic macrophages. The possibility that abnormally high levels of macrophage activation could predispose to dysregulations of the B cell lineage is raised.

Animals↗
PubMed1991

To B, or not to B: that is the question.

Irrespective of the prevailing fashion, Germinal Centre Conferences (GCC) have treated the immune system in a determinedly holistic manner. At the recent 10th GCC* the advantages of this approach were demonstrated in elegant new schema for lymphocyte development, accessory cell function, tolerance and autoimmunity, and for the organization and function of mucosal defence. This report concentrates on only one aspect of the meeting - the development of an integrated view of the natural history of B cells.

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PubMed1991

The effect of the graft-versus-host reaction on B lymphocyte production in bone marrow of mice. Depressed genesis of early progenitors prior to mu heavy chain expression.

The effects of systemic graft-versus-host (GVH) reactions on the early precursor cell populations involved in primary B lymphocyte genesis have been examined in the bone marrow of (C57BL/6xA)F1 mice injected with lymphoid cells from A strain mice. Double immunofluorescence labeling techniques for the intranuclear enzyme, terminal deoxynucleotidyl transferase (TdT), the B220 cell surface glycoprotein detected by monoclonal antibody, 14.8, and surface or cytoplasmic mu chains of IgM (s mu, c mu) were used to quantitate 3 putative early B lineage progenitors preceding mu chain expression (TdT+14.8-mu-, TdT+14.8+mu- and TdT-14.8+mu-), pre-B cells (c mu+, s mu-) and B lymphocytes (s mu+). After initiating GVH reactions, the early B precursor cells, pre-B cells, and B lymphocytes in the bone marrow all fell rapidly in numbers, being almost completely absent from 10-15 days to the end of the 30-day assay period. The decline of some of the early progenitors started at a later time and was less complete than that of the more differentiated B lineage cells. In the spleen, B lymphocytes declined rapidly in numbers after 8 days to less than 5% of normal values from 12 days onward. The results demonstrate that systemic GVH reactions in mice almost completely eliminate the B cell lineage, including early precursor cells apparently undergoing mu chain rearrangement in the bone marrow. The pattern of depletion suggests that a range of B lineage progenitor cells may be directly susceptible to GVH reactions. The findings contribute to a model for the pathogenesis of the humoral immunodeficiency of systemic GVH disease.

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PubMed1990

Microenvironmental organization and stromal cell associations of B lymphocyte precursor cells in mouse bone marrow.

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.

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PubMed1990

Proliferation of B cell precursors in bone marrow of pristane-conditioned and malaria-infected mice: implications for B cell oncogenesis.

Two widely different agents implicated in the etiology of neoplasias of the B cell lineage, pristane and malaria, have both been found to produce a prolonged increase in the level of proliferative activity and cell production by early B lymphocyte precursor cells in mouse bone marrow. This apparently leads to an elevated level of cell loss, suggesting the production of many aberrant early cells. The mechanism and significance of this effect remain to be determined. However, the present findings focus attention on the early stages of B cell genesis in the bone marrow as possible target cells for the initiation of genetic events leading to neoplasia. Together with previous work, the results suggest that pathologically elevated levels of macrophage activation may play a role in predisposing to various B cell neoplasias.

Animals↗
PubMed1990

B cell development in the bone marrow.

Recent years have produced considerable progress in defining stages in the development of B cells in vivo, and in revealing interactions with regulatory molecules and cells. Studies of the phenotype and population dynamics of precursor B cells in mouse bone marrow have quantitated cell production at sequential steps of differentiation and have also indicated a substantial cell death. The proliferation of precursor B cells is influenced both by systemic factors and by cytokines derived from bone marrow stromal cells. In situ immunolabeling has revealed that early precursor B cells are closely associated with subosteal stromal cells, aberrant B lineage cells appear to be deleted by macrophages and terminal B cells mature within the lumen of vascular sinusoids before being released. The findings lead to working models of the in vivo differentiation, regulation and microenvironmental organization of B cell genesis in the bone marrow.

Animals↗
PubMed1990

Early B-lymphocyte precursor cells in mouse bone marrow: subosteal localization of B220+ cells during postirradiation regeneration.

The localization of early B-lymphocyte precursor cells in the bone marrow of young mice has been studied during recovery from sublethal whole body gamma-irradiation (150 rad). Initial studies by double immunofluorescence labeling of the B-lineage-associated cell surface glycoprotein, B220, and of mu heavy chains in bone marrow cell suspensions, demonstrated a sequential wave of regeneration of early B precursor cells, pre-B cells, and B cells. Early B precursor cells expressing B220 but not mu chains were enriched at 1-3 days following irradiation. After in vivo administration of 125I-labeled monoclonal antibody 14.8 to detect B220+ cells in situ, light and electron microscope radioautography of femoral bone marrow sections revealed concentrations of labeled B220+ cells located peripherally near the cortical bone at 1-3 days following irradiation, increasing in numbers in more central areas by 5-7 days. Proliferative B220+ precursor cells were found within layers of bone-lining cells and in a subosteal area characterized by a prominent electron-dense extracellular matrix, often associated with stromal reticular cells. The results demonstrate that the precursor cells that are active in the bone marrow early in the recovery of B lymphopoiesis after gamma-irradiation are located both within and near the endosteum of the surrounding bone. The distinctive extracellular matrix and stromal cell associations noted in this region may contribute to a supportive local microenvironment for early hemopoietic progenitor cells.

Animals↗
PubMed1989

Dynamics of early B lymphocyte precursor cells in mouse bone marrow: proliferation of cells containing terminal deoxynucleotidyl transferase.

Three populations of early B lymphocyte precursor cells lacking mu heavy chains have been defined in mouse bone marrow, based on immunofluorescence labeling for terminal deoxynucleotidyl transferase (TdT) and B220 glycoprotein, as detected by monoclonal antibody 14.8 (TdT+14.8- cells; TdT+14.8+ cells; TdT-14.8+ cells). We have now analyzed the frequency, size distribution, proliferation and production rates of TdT+ cells in mouse bone marrow. These formed well-defined populations of medium-sized cells, the TdT+14.8+ cells tending to be larger than TdT+14.8- cells (modal cell diameters in cytocentrifuge preparations; 10.0 microns and 9.0 microns, respectively). Some TdT+ cells (1%-2%) were normally in metaphase, the TdT being dispersed through the cytoplasm. After inducing mitotic arrest with vincristine, the incidence of TdT+ cells in metaphase increased linearly from 2 to 4 h, indicating a turnover of 5.1%/h for TdT+14.8- cells and 9.0%/h for TdT+14.8+ cells. Subtraction of turnover data for TdT+14.8+ cells from those previously obtained for 14.8+ mu- cells gave values for the population of TdT-14.8+ cells. The calculated daily turnover of cells in the three compartments increased progressively (TdT+14.8-, 2.5 x 10(6) cells; TdT+14.8+, 5.0 x 10(6) cells; TdT-14.8+, 36.0 x 10(6) cells), accompanied by a shortening of the average apparent cell cycle time (TdT+14.8-, 20 h; TdT+14.8+, 11 h; TdT-14.8+, 8 h). The results demonstrate a progressive expansion of cell production at three putatively successive stages of early B lymphocyte development before the expression of mu chains. The findings contribute to a kinetic model of primary B cell genesis in mouse bone marrow.

Animals↗
PubMed1989

Post-irradiation regeneration of early B-lymphocyte precursor cells in mouse bone marrow.

To examine the sequential development of early B-cell precursors in mouse bone marrow, B-lineage cells have been examined during a wave of post-irradiation regeneration. Cell phenotypes have been defined using double-immunofluorescence labelling techniques for (i) terminal deoxynucleotidyl transferase (TdT); (ii) B220 glycoprotein, detected by the binding of mAb 14.8; (iii); mu heavy chains in the cytoplasm (c mu) and at the cell surface (s mu). Three populations of mu- cells (TdT+14.8-; TdT+14.8+; TdT-14.8+) have been proposed to be early B-cell precursors which would give rise to c mu+s mu- pre-B cells and thus to s mu+ B lymphocytes. From 3 to 7 days after a sublethal dose (150 rads) of whole body gamma-irradiation, the B-lineage cells recovered rapidly to exceed normal numbers. The early B-cell precursors increased to peaks of 1.8-2.5 times normal numbers, preceding by 1 day a comparable increase and overshoot of c mu+s mu- pre-B cells, followed by recovery of s mu+ B lymphocytes. The TdT+14.8- cells peaked first at 5 days, subsiding again at 7-10 days with a shift from large- to medium-sized cells. The TdT+14.8+ cells showed a later peak (6 days), a more sustained wave in cell numbers and a delayed shift in cell size. The substantial population of 14.8+ mu- cells reached maximal observed values at 7 days and still maintained a predominantly large cell size profile at 10 days. The timing, cell-size shifts and progressive amplification of the waves of regeneration accord with a dynamic model in which the TdT+14.8-,TdT+14.8+ and TdT-14.8+ cells form three successive stages in B-cell differentiation before the expression of mu chains, presumptively including the stage of mu chain gene rearrangement. In addition, the results provide an experimental system for the enrichment of early B-cell precursors in mouse bone marrow.

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PubMed1987

Phenotype and proliferation of early B lymphocyte precursor cells in mouse bone marrow.

Bone marrow cells were examined by double immunofluorescent labeling techniques to detect determinants for the B lineage monoclonal antibody, 14.8, the nuclear enzyme, terminal deoxynucleotidyl transferase (TdT), cytoplasmic mu chains (c mu), and surface mu (s mu). In 8-9-wk-old C3H/HeJ mice, 14.8+ cells totalled 22.2% of all marrow cells (35 X 10(5) cells/femur). While many 14.8+ cells were c mu+ s mu- pre-B cells and s mu+ B lymphocytes (17.0%), the remainder (5.2%) were large cells lacking mu chains. After injecting vincristine sulfate, these 14.8+ mu- cells accumulated in mitosis at a rate of 13.5%/h (turnover time, 7.4 h). Their calculated total production rate (41 X 10(6) cells/whole marrow/d) exceeded that previously determined for large pre-B cells, suggesting some cell loss from the B lineage. TdT+ cells made up 1.8% of marrow cells and were mainly medium-sized cells. They all lacked mu chains, but half (0.9%) bound 14.8 antibody at low to medium intensity. Three discrete cell populations were thus defined, differing in mean cell diameter TdT+ 14.8- mu-, 9.5 micron; TdT+ 14.8+ mu-, 10 microns; and TdT- 14.8+ mu-, 11.5 micron, presumptively representing a sequence of cell stages preceding the expression of mu chains in large pre-B cells (TdT- 14.8+ c mu+ s mu-, 11.5 microns). This work provides a tentative model of early progenitor cells and their proliferation in normal marrow as a basis for studies of perturbations and the control of B lymphocytopoiesis.

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