RESUMEN

Venous sinusoids in bone marrow are the site of a large-scale traffic of cells between the extravascular hemopoietic compartment and the blood stream. The wall of the sinusoids consists solely of a basement membrane interposed between a layer of endothelial cells and an incomplete covering of adventitial cells. To examine its possible structural specialization, the basement membrane of bone marrow sinusoids has now been examined by high resolution electron microscopy of perfusion-fixed mouse bone marrow. The basement membrane layer was discontinuous, consisting of irregular masses of amorphous material within a uniform 60-nm-wide space between apposing endothelial cells and adventitial cell processes. At maximal magnifications, the material was resolved as a random arrangement of components lacking the "cord network" formation seen in basement membranes elsewhere. Individual components exhibited distinctive ultrastructural features whose molecular identity has previously been established. By these morphological criteria, the basement membrane contained unusually abundant chondroitin sulfate proteoglycan (CSPG) revealed by 3-nm-wide "double tracks," and moderate amounts of both laminin as dense irregular coils and type IV collagen as 1-1.5-nm-wide filaments, together with less conspicuous amounts of amyloid P forming pentagonal frames. In contrast, 4.5-5-nm-wide "double tracks" characteristic of heparan sulfate proteoglycan (HSPG) were absent. The findings demonstrate that, in comparison with "typical" basement membranes in other tissues, the bone marrow sinusoidal basement membrane is uniquely specialized in several respects. Its discontinuous nature, lack of network organization, and absence of HSPG, a molecule that normally helps to maintain membrane integrity, may facilitate disassembly and reassembly of basement membrane material in concert with movements of adventitial cell processes as maturing hemopoietic cells pass through the sinusoidal wall: the exceptionally large quantity of CSPG may represent a reservoir of CD44 receptor for use in hemopoiesis.

Asunto(s)

Médula Ósea/química , Médula Ósea/ultraestructura , Fémur/química , Fémur/ultraestructura , Proteoglicanos/análisis , Animales , Membrana Basal/química , Espacio Extracelular , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica

RESUMEN

Mice transgenic for a p190bcr/abl construct develop pre-B cell leukemia/lymphoma, providing a model of Ph+ ALL. To investigate events in tumorigenesis, immunofluorescence labeling, flow cytometry and a short-term culture assay were used to quantitate precursor B cells and their apoptotic rates in bone marrow of p190bcr/abl transgenic mice over a wide age range. Malignancies appeared rapidly at 8-12 weeks of age, followed by slower tumor onset. At 8-12 weeks in normal mice, the apoptotic rate fell among pro-B cells but increased steeply among pre-B cells, while the total number of B lineage cells declined. In contrast, in p190bcr/abl transgenic mice over the same time period, while pro-B cells remained normal in apoptotic rate and number, apoptosis of pre-B cells was markedly inhibited and the number of B lymphocytes increased. At later ages (14-30 weeks), B cell precursors in control mice remained constant in apoptotic activity and number, while in the few surviving transgenic mice B cell populations were expanded. The results reveal characteristic changes in apoptotic activity among B cell precursors in bone marrow during early life, severely perturbed in preleukemic p190bcr/abl transgenic mice by a preferential suppression of pre-B cell apoptosis. p190bcr/abl may thus promote leukemogenesis by permitting aberrant cells generated during early B cell development to evade a normal quality checkpoint and negative selection.

Asunto(s)

Apoptosis , Linfocitos B/fisiología , Células de la Médula Ósea/fisiología , Proteínas de Fusión bcr-abl/fisiología , Células Madre Hematopoyéticas/fisiología , Preleucemia/patología , Factores de Edad , Animales , Daño del ADN , Ratones , Ratones Transgénicos

RESUMEN

OBJECTIVE: Osteopetrotic (op/op) mice are deficient in macrophages and osteoclasts due to a CSF-1 gene mutation. The aim of this study was to evaluate the effect of these deficiencies and of CSF-1-dependent mechanisms on B lymphopoiesis in bone marrow, with special reference to the apoptotic activity of precursor B cells. MATERIALS AND METHODS: B-cell development and apoptosis were examined in the bone marrow of op/op mice using immunofluorescence labeling and flow cytometry. Short-term cultures of bone marrow were used to evaluate the effect of recombinant CSF-1 on the rate of B-cell apoptosis. RESULTS: Bone marrow cellularity was greatly reduced in op/op mice compared with normal littermates. However, precursor B cells were disproportionately decreased, most markedly at the pre-B-cell stage. Precursor B cells, particularly pre-B cells, displayed elevated apoptotic incidences both ex vivo and in short-term culture. Addition of recombinant CSF-1 reduced the incidence of apoptosis among precursor B cells in short-term cultures of whole bone marrow suspensions from normal mice but not in cultures of sorted B220+ B-lineage cells. CONCLUSIONS: The finding of increased pre-B-cell apoptosis in op/op mice provides evidence that CSF-1-dependent mechanisms can strongly influence the survival of precursor B cells in mouse bone marrow, particularly at the pro-B/pre-B cell transition. It is proposed that the local or systemic levels of CSF-1 during ontogeny may thus play a role in regulating B-cell production within the bone marrow microenvironment.

Asunto(s)

Apoptosis , Linfocitos B/patología , Médula Ósea/patología , Células Madre Hematopoyéticas/patología , Factor Estimulante de Colonias de Macrófagos/deficiencia , Osteopetrosis/patología , Animales , Apoptosis/efectos de los fármacos , Linaje de la Célula , Células Cultivadas/efectos de los fármacos , Femenino , Hematopoyesis , Células Madre Hematopoyéticas/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/farmacología , Masculino , Ratones , Ratones Mutantes , Receptor de Factor Estimulante de Colonias de Macrófagos/análisis , Proteínas Recombinantes/farmacología

RESUMEN

CD24 (heat-stable antigen) is expressed in a developmentally regulated fashion by B cell precursors in mouse bone marrow (BM), but its role in B lymphopoiesis remains obscure. A slight overexpression of CD24 in transgenic (Tg) mice leads to depletion of B lymphoid cells in BM. The present study examines whether CD24 is involved in apoptotic selection of B lineage cells under normal microenvironmental conditions in vivo. Double immunofluorescence labeling and flow cytometry have been used to quantitate the apoptotic rates of phenotypically defined B cell populations in BM of CD24-Tg mice. Apoptosis of pre-B cells expressing cytoplasmic mu heavy chains of IgM but lacking surface (s)IgM was increased both ex vivo and in short-term culture, while the number of pre-B cells was halved compared to BM of normal mice. In contrast, B220+mu- pro-B cells and sIgM+ B lymphocytes showed no significant change in either apoptosis or number. The findings provide evidence that CD24 can play a role in vivo in modulating pre-B cell apoptosis, a quality control checkpoint in B cell development.

Asunto(s)

Antígenos CD/fisiología , Apoptosis , Linfocitos B/fisiología , Células de la Médula Ósea/fisiología , Hematopoyesis , Células Madre Hematopoyéticas/fisiología , Glicoproteínas de Membrana , Animales , Antígeno CD24 , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos

RESUMEN

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 lambda5 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.

Asunto(s)

Apoptosis , Linfocitos B/fisiología , Células de la Médula Ósea/fisiología , Melatonina/metabolismo , Células Madre/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Células Cultivadas , ADN/metabolismo , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inmunofenotipificación , Melatonina/farmacología , Ratones , Ratones Endogámicos C3H , Células Madre/citología , Células Madre/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Apoptosis/fisiología , Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Glicoproteínas de Membrana , Animales , Antígenos CD/genética , Antígenos CD/fisiología , Antígeno CD24 , Diferenciación Celular , Linaje de la Célula , Factores Estimulantes de Colonias/genética , Factores Estimulantes de Colonias/fisiología , Proteínas de Unión al ADN/genética , Homeostasis , Interleucina-7/genética , Interleucina-7/fisiología , Ratones , Ratones Mutantes , Modelos Biológicos , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/fisiología , Receptores de Antígenos de Linfocitos B/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiología

RESUMEN

Multinucleated giant cells have been observed at interfaces between bone marrow and titanium implants in mouse femurs. This raises concern that macrophage-derived factors might perturb local lymphohemopoiesis, possibly even predisposing to neoplasia in the B lymphocyte lineage. It has been found that an implant-marrow interface with associated giant cells persists for at least 1.5 years. Precursor B cells show early increases in number and proliferative activity. At later intervals, however, they do not differ significantly from controls, and there are no perturbations in spatial localization of either B lineage cells or DNA-synthesizing hemopoietic cells. The results of this investigation in mice demonstrate that, following initial marrow regeneration and fluctuating precursor B cell activity, and despite the presence of giant cells, titanium implants apparently become well-tolerated by directly apposed bone marrow cells in a lasting state of "myelointegration."

Asunto(s)

Linfocitos B/fisiología , Materiales Biocompatibles , Células de la Médula Ósea/fisiología , Implantes Dentales , Células Madre Hematopoyéticas/fisiología , Leucopoyesis/fisiología , Oseointegración , Titanio , Análisis de Varianza , Animales , Materiales Biocompatibles/química , Neoplasias Óseas/etiología , Regeneración Ósea/fisiología , Recuento de Células , División Celular , Linaje de la Célula , Susceptibilidad a Enfermedades , Fémur/patología , Células Gigantes/fisiología , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos , Propiedades de Superficie , Factores de Tiempo , Titanio/química

RESUMEN

B cell development in mouse bone marrow (BM) is subject to quality controls that eliminate aberrant cells by apoptosis, but the intrinsic cellular mechanisms that mediate this negative B cell selection remain unclear. The p53 tumor suppressor transduces signals resulting in apoptosis and cell cycle arrest in cells that sustain DNA damage. Faulty V(D)J recombination in scid lymphocyte precursors activates a p53-dependent DNA damage checkpoint. In the present study, we have examined whether p53 is involved in apoptotic selection of normally developing B cells in BM. Double immunofluorescence labeling and flow cytometry were used to quantitate phenotypically defined B cell populations and their apoptotic rates in BM of homozygous p53-deficient mice. B220(+) mu(-) and terminal deoxynucleotidyl transferase (TdT)(+) pro-B cells were increased in both incidence and absolute number to controls. In contrast, pre-B cells were only slightly increased and the sIgM(+) B lymphocyte compartment remained essentially normal. The incidence of apoptosis among p53(-/-) pro-B cells was greatly reduced, both ex vivo and in short-term culture, whereas, apoptosis of pre-B cells and B lymphocytes was not significantly different from normal. The results indicate that p53 is actively involved as an apoptosis inducer at an early quality control checkpoint in B lymphopoiesis.

Asunto(s)

Linfocitos B/citología , Linfocitos B/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Animales , Apoptosis , Linfocitos B/inmunología , Diferenciación Celular , Supervivencia Celular , Genes p53 , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Técnicas In Vitro , Ratones , Ratones Noqueados , Proteína p53 Supresora de Tumor/genética

RESUMEN

B cell development in mouse bone marrow depends critically upon IL-7. To examine the possible in vivo trophic role of IL-7, we have quantitated apoptosis and Bcl-2 family proteins in populations of phenotypically defined B lineage cells in IL-7-deficient and IL-7-overexpressing mice. Using immunofluorescence labeling, multiparameter flow cytometry, and a short-term culture assay, we show that the apoptotic rates of precursor B cells, but not of more mature B cells, are enhanced by IL-7 gene deletion, associated with increased intracellular content of Bax and decreased Bcl-2, while, conversely, an IL-7 transgene suppresses precursor B cell apoptosis and produces low Bax and high Bcl-2 levels. During normal B cell development, high Bax/Bcl-2 ratios characterize cells undergoing greatest apoptotic cell death. Pro-B cells in RAG-2-/- mice, all destined to abort, show elevated Bax levels and Bax/Bcl-2 ratios. By comparison with the elevated rate of pro-B cell apoptosis in RAG-2-/- mice, provisional estimates have been made for the fraction of pro-B cells undergoing apoptosis in normal mice (70%), IL-7-/- mice (85%), and IL-7 transgenic mice (35%). The results demonstrate that IL-7 strongly promotes in vivo cell survival and maintains antiapoptotic Bcl-2/Bax ratios during the development of precursor B cells in mouse bone marrow.

Asunto(s)

Apoptosis/inmunología , Linfocitos B/citología , Células de la Médula Ósea/citología , Proteínas de Unión al ADN/genética , Células Madre Hematopoyéticas/citología , Interleucina-7/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis/genética , Linfocitos B/metabolismo , Células de la Médula Ósea/metabolismo , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Proteínas de Unión al ADN/biosíntesis , Hematopoyesis/genética , Hematopoyesis/inmunología , Células Madre Hematopoyéticas/metabolismo , Interleucina-7/biosíntesis , Interleucina-7/deficiencia , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Transposasas/genética , Proteína X Asociada a bcl-2

RESUMEN

Many B cell precursors die while differentiating in mouse bone marrow. To ascertain the mechanisms involved in this process, populations of B lineage cells and their tissue localization were analyzed in bone marrow of transgenic mice overexpressing the apoptosis inhibitor, Bcl-2. Immunofluorescence labeling and mitotic arrest were used to quantitate the number and proliferative activity of mu- pro-B cells (terminal deoxynucleotidyl transferase [TdT]+B220-, TdT+B220+, and TdT-B220+); pre-B cells (cmu+); and B cells (smu+). Mature B cells (IgM+IgD+) were increased 16- to 20-fold. In addition, immature B lymphocytes (IgM+IgD-/low), representing newly formed cells, were increased three- to sixfold, whereas pre-B cells and late pro-B cells were increased 30 to 60% in production rate. Earlier pro-B cells expressing TdT were unaffected. In spleen, both mature and immature B cells were greatly increased, but cells of precursor phenotype were few and TdT+ cells were absent. The in vivo location of B cells was examined by autoradiography using light and electron microscopy after intravenous injection of 125I-labeled antibodies. B lineage cells (B220+) were increased throughout bone marrow, often within dilated venous sinusoids, particularly in subosteal regions. Many intravascular and perisinusoidal cells were IgDhigh mature B lymphocytes. In contrast, many other IgM+ and IgDlow immature B lymphocytes clustered extravascularly around the central venous sinus. Plasma cells with distended endoplasmic reticulum were numerous. These findings provide evidence that, in addition to expanding the recirculating pool of B cells entering bone marrow from the blood stream, high levels of Bcl-2 can inhibit some of the apoptosis occurring during B cell differentiation, thereby expanding populations of B lymphopoietic precursor cells within the bone marrow parenchyma.

Asunto(s)

Apoptosis/genética , Linfocitos B/inmunología , Médula Ósea/inmunología , Genes bcl-2 , Animales , Diferenciación Celular/inmunología , Linaje de la Célula , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre/inmunología

RESUMEN

B lymphocyte development in mouse bone marrow (BM) occurs in association with stromal cells which provide essential growth factors, notably interleukin 7 (IL-7). The ability of recombinant IL-7 given systemically by several modes of administration to stimulate proliferation of precursor B cells at successive stages of development was examined. Using immunofluorescence labelling and mitotic arrest, the in vivo proliferative cell dynamics of phenotypically defined B lineage cells was quantitated. Single injections of murine IL-7 (muIL-7) in low and intermediate doses stimulated production both of pro-B cells preceeding the expression of mu heavy chains and large pre-B cells expressing cytoplasmic mu. In contrast, higher doses were not stimulatory. Infusion of muIL-7 from subcutaneous micro-osmotic pumps produced a proliferative wave of TdT+ pro-B cells and pre-B cells followed by elevated numbers of postmitotic small pre-B cells and B cells. Stimulation tended to be followed by secondary oscillations of B lymphopoiesis or an IL-7 refractory state. Pronounced increases in TdT+ pro-B and pre-B cell production resulted from injecting small doses of either muIL-7 or human IL-7 (hIL-7), complexed with anti-IL-7 antibody as carrier protein. The results indicate that exogenous IL-7 at optimal dose can markedly stimulate in vivo B lymphopoiesis from the earliest detectable TdT+ pro-B cell stage onward, and is effective when delivered as a cytokine-anticytokine complex.

Asunto(s)

Linfocitos B/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Interleucina-7/administración & dosificación , Animales , Complejo Antígeno-Anticuerpo/farmacología , Linfocitos B/citología , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Infusiones Parenterales , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Interleucina-7/inmunología , Masculino , Ratones , Ratones Endogámicos C3H , Microscopía Fluorescente , Proteínas Recombinantes/farmacología , Bazo/citología , Bazo/efectos de los fármacos

RESUMEN

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 mu heavy chain of IgM (c mu) 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 c mu 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.

Asunto(s)

Linfocitos B/citología , Leucopoyesis , Modelos Inmunológicos , Animales , Linfocitos B/metabolismo , Células de la Médula Ósea , Diferenciación Celular , Separación Celular , ADN Nucleotidilexotransferasa/biosíntesis , Citometría de Flujo , Cadenas mu de Inmunoglobulina/biosíntesis , Inmunofenotipificación , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente

Asunto(s)

Linfocitos B/citología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores , Médula Ósea/inmunología , División Celular/inmunología , Hematopoyesis/inmunología , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Ratones , Modelos Inmunológicos

RESUMEN

The purpose of this study was to use amelogenin as a marker to examine the feasibility of isolating ameloblasts from enamel organ cell populations by fluorescence activated cell sorting. After treating dissected rat enamel organs with proteolytic enzymes to loosen cell attachments and labial connective tissues, dissociated cell suspensions were fixed, then immunostained with rabbit anti-rM179 recombinant amelogenin antibody and FITC-conjugated goat anti-rabbit Ig G antibody. Flow cytometry indicated that about 70% of the total cell sample and virtually all the larger cells therein were amelogenin-positive. Fluorescence activated cell sorting yielded a sample of amelogenin-positive cells at 97% purity. Immunofluorescence microscopy indicated that these isolated amelogenin-positive cells varied widely in size and morphology. This was attributed to loss of intercellular support for ameloblasts once they were dissociated from each other, and to some fragmentation caused when the cells were initially physically removed from the teeth. The results demonstrate that viable ameloblast cell fractions, especially representing cells at the secretory stage, can be purified from enzymic digests of rat enamel organ by sorting on the basis of cell size alone. From these fractions, subpopulations of ameloblasts may be identified when differentiation specific cell surface markers become available.

Asunto(s)

Ameloblastos/citología , Incisivo/citología , Ameloblastos/metabolismo , Amelogenina , Animales , Separación Celular/métodos , Proteínas del Esmalte Dental/inmunología , Proteínas del Esmalte Dental/metabolismo , Pulpa Dental/citología , Órgano del Esmalte/citología , Órgano del Esmalte/metabolismo , Citometría de Flujo/métodos , Fluorescencia , Técnica del Anticuerpo Fluorescente Indirecta , Mandíbula , Microscopía Fluorescente , Conejos , Ratas , Ratas Sprague-Dawley , Coloración y Etiquetado

RESUMEN

To evaluate the magnitude of cell death and the critical stages at which it occurs during B lymphopoiesis in mouse bone marrow (BM), we have examined the kinetics of apoptosis at defined stages of B cell differentiation. FACS-sorted B220+ BM cells exhibited a low incidence of morphologically apoptotic cells by electron microscopy. In freshly prepared BM suspensions, the incidence of hypodiploid cells detected by multiparameter flow cytometry was greater among large dividing B220+ surface IgM- (sIgM-) precursor B cells and sIgM(low) immature B lymphocytes than among terminal deoxynucleotidyl transferase+ (TdT+) pro-B cells, small nondividing B220+ sIgM- precursors, and surface IgD+ mature B lymphocytes. During short-term culture, apoptotic cells, identified by both DNA content and in situ DNA strand break labeling, increased linearly with time without macrophage ingestion, providing an assay for the rate of entry into apoptosis. B220+ B lineage cells accumulated in apoptosis more rapidly than cells of other lineages. The apoptotic rate was greater among B220+ sIgM- precursor cells than sIgM+ B cells, and was highest among B220+ mu- pro-B cells. Coculture with stromal cells reduced the apoptotic rate of B220+ sIgM- precursors to a greater extent than that of sIgM+ B lymphocytes. The results lead to estimates of the actual number of B lineage cells undergoing apoptosis per unit time in successive differentiation compartments. The findings indicate that, although influenced by local microenvironmental factors, apoptotic cell death occurs most markedly at two developmental stages associated with Ig heavy chain gene rearrangement and Ag receptor expression, respectively.

Asunto(s)

Apoptosis , Linfocitos B/patología , Médula Ósea/patología , Animales , Linfocitos B/inmunología , Médula Ósea/inmunología , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Masculino , Ratones , Ratones Endogámicos C3H , Células del Estroma/patología

RESUMEN

E(mu)-myc transgenic mice carry a constitutively overexpressed c-myc oncogene and develop B-lineage lymphomas. Previous studies have shown that c-myc overexpression can lead to in vitro apoptosis. Here, we investigated the in vivo effects of altered c-myc expression on cell proliferation versus death in spontaneously arising E(mu)-myc tumors. E(mu)-myc tumors display extensive in vivo apoptosis confined to small clusters of cells with greatly increased expression of both the c-myc transgene and the endogenous p53 gene as compared with that in normal, pretumor, or surrounding tumor tissue. This restricted overexpression of both the c-myc transgene and the endogenous p53 gene in small clusters of apoptotic tumor cells indicates that overexpression of these genes and apoptosis are not obligatory or uniform during tumor development and suggests that further somatic mutations or microenvironmental influences may be responsible for these properties. Nevertheless, the clear ability of tumor cells to undergo apoptosis in vivo may be exploitable for therapeutic purposes.

Asunto(s)

Apoptosis/genética , Regulación Neoplásica de la Expresión Génica , Genes myc , Genes p53 , Linfoma de Células B/genética , Regulación hacia Arriba/genética , Animales , ADN de Neoplasias/análisis , Citometría de Flujo , Inmunohistoquímica , Tejido Linfoide/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena de la Polimerasa

RESUMEN

Transgenic mice carrying mouse interleukin-7 (IL-7) cDNA under the control of MHC class II (E alpha) promoter develop B lymphoid tumors. We have analyzed population dynamics of early precursor B cells and electron microscopic organization of bone marrow (BM) during the prelymphomatous phase. Immunofluorescence labeling of terminal deoxynucleotidyl transferase (TdT), B220 glycoprotein, and mu heavy chains have been used to quantitate three populations of pro-B cells lacking mu chains, cytoplasmic mu-bearing pre-B cells, and surface mu-bearing B lymphocytes. Proliferative activity was assayed by metaphase arrest. In BM of IL-7 transgenic mice, the number and proliferative activity of cells in each of the pro-B and pre-B cell populations were markedly increased. B lymphocytes increased to a lesser extent. The BM cavity was considerably expanded and cortical bone showed focal osteolysis. Immature lymphoid cells compressed the venous sinusoids and exuded through eroded bone. Apoptotic bodies, macrophages, and plasma cells were unusually prominent. B lymphocytes and cells of B precursor phenotype were also much increased in the spleen. These results demonstrate that overexpression of IL-7 causes excessive proliferation of a wide range of precursor B cells in BM. Such prolonged stimulation at early stages of B cell development, prone to genetic errors, may predispose to neoplasia. The bone resorption in these transgenic mice provides a model for bone lesions in BM malignancies.

Asunto(s)

Linfocitos B/citología , Células de la Médula Ósea , Interleucina-7/genética , Interleucina-7/inmunología , Linfoma de Células B/patología , Ratones Transgénicos/genética , Ratones Transgénicos/inmunología , Animales , Linfocitos B/inmunología , División Celular/inmunología , ADN Complementario/análisis , Femenino , Técnica del Anticuerpo Fluorescente , Cadenas Pesadas de Inmunoglobulina/análisis , Cadenas mu de Inmunoglobulina/análisis , Masculino , Ratones , Mitosis , Bazo/citología

RESUMEN

B lymphopoiesis in mouse bone marrow (BM) can be stimulated by circulating products derived from activated macrophages in the spleen. To examine whether IL-1 could mediate this effect, we have administered murine rIL-1alpha in a range of doses, determining its effect on precursor B cells and its capacity to bind to stromal cells in BM. Immunofluorescence labeling of terminal deoxynucleotidyl transferase (TdT), B220 glycoprotein, and mu-chains has been used to quantitate pro-B cells lacking mu (TdT+; 13220+mu-), pre-B cells expressing cytoplasmic mu, and B lymphocytes bearing surface mu. Proliferative activity was measured by mitotic arrest. Single i.p. injections of rIL-1alpha produced a proliferative stimulation of pro-B cells and pre-B cells at optimal doses, whereas other doses were suppressive. Infusion of rIL-1alpha from s.c. osmotic pumps depressed B lymphopoiesis at high dose rates, but stimulated precursor B cell proliferation at lower dose rates. Intravenous 125I-labeled rIL-1alpha bound strongly to a subset of stromal reticular cells and sinusoidal endothelium in BM, as detected by light and electron microscope radio-autography. Computer-aided analysis located rIL-1alpha-binding stromal cells mainly in the outer zones of BM, sites of proliferating precursor B cells, rather than the more central zone. The results demonstrate that IL-1 can act systemically at various dose levels as either a positive or negative modifier of B lymphopoiesis in BM, probably acting indirectly via stromal reticular cells and endothelial cells. Thus, inflammatory processes associated with macrophage activation and IL-1 secretion may have pronounced effects on B cell genesis in BM.

Asunto(s)

Linfocitos B/inmunología , Médula Ósea/inmunología , Médula Ósea/metabolismo , Hematopoyesis/inmunología , Interleucina-1/metabolismo , Interleucina-1/farmacología , Animales , Linfocitos B/metabolismo , Linfocitos B/ultraestructura , Sitios de Unión , Células de la Médula Ósea , Relación Dosis-Respuesta Inmunológica , Endotelio/citología , Endotelio/inmunología , Endotelio/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Interleucina-1/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C3H , Microscopía Electrónica , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología

RESUMEN

Cell adhesion molecules (CAMs) play a key role in interactions between stromal and hematopoietic cells in bone marrow (BM) and in cell traffic through vascular endothelium. To examine the identity of CAMs involved in these processes in mouse BM, we have investigated the in vivo expression of vascular cell adhesion molecule-1 (VCAM-1) and its counter-receptor, very late antigen-4 (VLA-4). Radioiodinated monoclonal antibodies (MoAbs) detecting VLA-4 and VCAM-1 were injected intravenously. Antibody binding was detected in BM by light and electron microscope radioautography. VCAM-1 labeling was restricted to stromal reticular cells and endothelial cells lining BM sinusoids. VCAM-1+ reticular cells formed patchy concentrations, especially in subosteal regions, associated with lymphoid, granulocytic, and erythroid cells. After gamma-irradiation to deplete hematopoietic cells, reticular cells and endothelial cells all showed VCAM-1 labeling in apparently increased intensity. VLA-4 labeling was shown by undifferentiated blast cells and lymphohematopoietic cells both in BM cell suspensions and in vivo, especially at reticular cell contact points. The results demonstrate that VCAM-1 is expressed in vivo by certain BM reticular cells, suggesting that the molecule mediates adhesion to multiple lineages of lymphohematopoietic cells. The finding that VCAM-1 is also expressed constitutively by BM sinusoidal endothelium, unlike its inductive expression by endothelia elsewhere, suggests that VCAM-1 and VLA-4 may be involved in regulating the normal cell traffic between BM and the blood stream.

Asunto(s)

Anemia Aplásica/patología , Células de la Médula Ósea , Células del Tejido Conectivo , Endotelio Vascular/citología , Regulación de la Expresión Génica , Traumatismos Experimentales por Radiación/patología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Irradiación Corporal Total , Anemia Aplásica/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Médula Ósea/irrigación sanguínea , Médula Ósea/efectos de la radiación , Adhesión Celular , Movimiento Celular , Tejido Conectivo/metabolismo , Tejido Conectivo/efectos de la radiación , Endotelio Vascular/metabolismo , Endotelio Vascular/efectos de la radiación , Rayos gamma , Células Madre Hematopoyéticas/citología , Integrina alfa4beta1 , Integrinas/biosíntesis , Integrinas/genética , Integrinas/inmunología , Masculino , Ratones , Ratones Endogámicos C3H , Traumatismos Experimentales por Radiación/metabolismo , Receptores Mensajeros de Linfocitos/biosíntesis , Receptores Mensajeros de Linfocitos/genética , Receptores Mensajeros de Linfocitos/inmunología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunología

RESUMEN

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.

Asunto(s)

Linfocitos B/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Plasmacitoma/inducido químicamente , Terpenos/toxicidad , Animales , Linfocitos B/patología , Médula Ósea/patología , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Plasmacitoma/patología , Especificidad de la Especie , Bazo/efectos de los fármacos , Bazo/patología