Evidence of oxidative injury during aging of the liver in a mouse model.

The aim of the present study was to determine whether oxidative stress contributes to aging of the liver in a mouse model. Liver was obtained from young (3-5 months old) and aged (18-24 months old) mice. No age-induced gross changes in liver morphology were detected by light microscopy. Apoptosis was measured using the fragment end labeling of DNA for the immunohistochemical identification of the apoptotic nuclei. The total apoptotic cells represented 1% of the total cells in livers of young mice and 8% in those of aged mice. Among the total apoptotic cells in livers of aged animals, 15% were hepatocytes, 40% sinusoidal endothelial cells, and 45% bile duct cells. Hepatic lipid peroxidation, expressed as malonaldehyde levels, protein oxidation, measured by protein carbonyl content, and DNA oxidation, measured as 8-hydroxy-2'-deoxyguanosine (oxo(8)dG), were significantly increased in the livers of aged animals as compared to younger mice. The apoptotic cells presented elevated levels of oxidized DNA, detected by immunohistochemistry using an antibody directed against oxo(8)dG in serial sections. These results suggest that livers of aged animals presents evidence of increased oxidative injury and apoptosis. Because the apoptotic cells in the aged livers are mostly bile duct cells and sinusoidal endothelial cells, the cells most sensitive to oxidative stress injury, it can be hypothesized that reactive oxygen species-induced apoptosis in these cells contributes to the aging of the liver.

Impaired ability of bone marrow stromal cells to support B-lymphopoiesis with age.

B-lymphopoiesis decreases with age. We studied how aging affects bone marrow stromal cells, because they provide the growth factors and cell contacts required for B-lymphopoiesis. No differences were noted in the cell-surface phenotype of young and old primary-cultured stromal cells. Fluorescence-activated cell sorter-purified stromal cells from old mice were deficient in the ability to support the proliferation of interleukin-7 (IL-7)-specific B-lymphoid cell lines. The kinetics of this response indicated that IL-7 was not immediately available from stromal cells of either age and was further delayed on aged stromal cells. The levels of IL-7 protein within stromal cells were equivalent between young and old animals, suggesting that the production of IL-7 was not altered by aging. Negligible amounts of IL-7 were found either freely secreted or in the extracellular matrix of cultures of young and old marrow. Contact between the lymphoid cells and the primary stromal cells was required for detectable proliferation, suggesting that cell contact was required for the release of IL-7. We propose that stromal cells regulate B-lymphopoiesis by limiting the amount of IL-7 available to the developing precursors. Therefore, we conclude that the age-related decrease in the function of bone marrow stromal cells is related to the impaired release of IL-7.

Gamma 2b provides only some of the signals normally given via mu in B cell development.

B cell development is a complex process involving interactions between B cell precursors, stroma, and known and unknown ligands and cytokines. In order to more fully understand the requirements for Ig in that development we have created transgenic mice that carry a gamma 2b transgene and express it early in B cell development. Previously it was believed that these B cells arrested in their development prior to the pro- to pre-B cell transition. We show here that in conventional gamma 2b mice, B cell development actually arrests later, at the pre-B cell stage. This shows for the first time that a constant region different from mu can allow signaling through the pre-B cell receptor, but cannot promote complete development. The pro- and pre-B cells in the conventional gamma 2b transgenics are not fully functional since they cannot grow in IL-7 without stromal cells. This is a novel phenotype, separating development from stroma independence. The few, mature B cells that do develop in these mice express both mu and gamma 2b simultaneously, and are CD5+. Expression of a Bcl-2 transgene allows survival of gamma 2b transgenic immature B cells, but does not promote full maturation, indicating that normally mu provides both an anti-apoptotic signal and a differentiation signal. One line of gamma 2b mice, the C line, does not have this phenotype. B cell development is accelerated in this unconventional line, and the developing B cells have a very different phenotype from both normal mice and conventional gamma 2b mouse lines, but are very similar to mu transgenics. Mature B cells are largely CD5-, gamma 2b-only expressing. This unique phenotype is apparently due to the activation in B cell precursors of a gene at the insertion site of the transgene, circumventing the need for mu. Comparison of conventional gamma 2b transgenics with the C line and mu transgenics reveals the multiple signals required throughout B cell development.

Development of B cells in aged mice: decline in the ability of pro-B cells to respond to IL-7 but not to other growth factors.

The process of B lymphopoiesis changes with increasing age, resulting in a severe drop in the number of pre-B cells in old mice. We have shown that the ability of freshly isolated pro-B cells to proliferate on stromal cells declines with age. In this study, we wanted to determine the reason for the diminished response of the aged pro-B cells. The functional alterations could arise from changes intrinsic to the pro-B cells or to composition of the precursor population. Changes in the composition of pro-B cells in marrow or long-term bone marrow culture system for B lymphocytes (LTBMC-B) could not account for the functional losses. We then examined the hypothesis that the diminished amount of proliferation on stromal cells was due to changes in the responsiveness to stroma-derived cytokines. The proliferation to IL-7 but not to stem cell factor (SCF) or insulin-like growth factor 1 (IGF-1) was severely impaired by 24 mo of age, independently of the concentration of IL-7 and length of culture time. The reduced IL-7 response could not be explained by an increase in the percentage of cells undergoing apoptosis; rather, a greater frequency of aged cells remained in G0/G1 after IL-7 stimulation. Furthermore, the impaired responsiveness of the aged pro-B cells is not due to diminished expression of the IL-7R alpha-chain or the common gamma-chain. Since only IL-7 responsiveness is impaired, we believe that the underlying molecular mechanism for the cellular alterations is specific to signaling through the IL-7 receptor complex.

Regulation of IgM and IgD heavy chain gene expression: effect of abrogation of intergenic transcriptional termination.

Early IgM+ B cells express little or no membrane IgD due to the low abundance of delta mRNA. Extensive transcriptional termination regulated by sequences in the intronic region between mu and delta heavy chain genes may be the primary reason for the lack of delta gene transcription. We have examined the effect of deletion of these sequences on the regulation of IgM and IgD heavy chain gene expression in transfectants as well as mice carrying this otherwise intact transgene. By run-on transcriptional measurement, we show that the delta exons are transcribed in bone marrow B cells from these transgenic mice. However, in spite of the induced premature synthesis of the full-length mu-delta transcript in pre-B cells, processing to delta mRNA does not occur until the lymphocytes express cell surface IgM. Therefore, during B cell development, synthesis of the full-length transcript is a necessary but not sufficient condition for initiation of delta mRNA synthesis. Furthermore, unexpectedly, the abrogation of transcriptional termination was found to also affect the processing of the primary transcript to microM mRNA. These results show that expression of IgD in primary B cells is stringently regulated and closely linked to IgM expression.

Estrogen suppression of connective tissue deposition in a murine model of peritoneal adhesion formation.

Estrogen's involvement in inflammation and wound healing is poorly understood. To examine the role of estrogen in peritoneal adhesion formation, we gave ovariectomized female C57BL/6 mice time-release pellets containing placebo, 0.05 mg 17 beta-estradiol (low E2), or 5 mg 17 beta-estradiol (high E2) before i.p. injection of talc in saline or saline alone. Analyses of abdominal wall connective tissue thickness and peritoneal cell populations were performed. Talc-treated mice receiving low and high E2 replacement had a decreased amount of abdominal connective tissue deposition (29% and 65% decrease, respectively) as compared with talc-treated mice receiving placebo pellets. At high E2 replacement, the difference in connective tissue deposition was significant statistically (p less than 0.01). Immunohistochemical analysis revealed that the number of macrophages in adhesion tissue was proportionate to the amount of connective tissue present, regardless of the circulating levels of E2. Northern blot analysis of abdominal wall tissue showed that five of six talc-treated animals given placebo expressed mRNA for the murine monocyte chemoattractant protein-1 (MCP-1), JE. Conversely, only one of five talc-treated animals that received E2 replacement expressed JE/MCP-1 mRNA, suggesting that the hormone may inhibit connective tissue deposition by altering the production of chemotactic factors. Furthermore, E2 suppressed talc-induced expression of JE/MCP-1 mRNA in murine macrophages. Since macrophages play a central role in the wound healing process, these studies suggest that E2 inhibition of adhesion formation could be mediated by suppressing macrophage activation and/or recruitment to inflammatory sites.

Stage-specific alterations in murine B lymphopoiesis with age.

Although it is reported that B lymphopoiesis declines with age, the precursor stage(s) affected and the age of onset are ambiguous. Each progressive phase of B cell differentiation has distinct requirements; therefore, precise identification of the stage(s) that decline would yield insight into the age-related mechanisms affecting humoral immunity. We analysed the composition of B lineage cells of mice 1, 4, 12 and 24 months of age using flow cytometry. Numbers of prepro-B and pro-B cells were unchanged, and a profound decrease occurred only in the numbers of pre-B cells. This decrease occurred in two distinct phases: between 1 and 4 months and between 12 and 24 months. Notably, the numbers of newly formed B cells did not decline in parallel, suggesting that mechanisms are established to overcome the deficiency of pre-B cells. Since the age-related changes are limited to the pre-B cell stage, we hypothesized that the impairment acts at the pro-B to pre-B transition. We therefore evaluated whether the pro-B cells or the supporting stromal cells, which are necessary for normal progression of this stage, changed with age. The ability of pro-B cells to proliferate in the presence of stromal cells was reduced by 24 months of age, as was the ability of the stromal cells to support pro-B cell proliferation. In contrast, the ability to mature into IgM+ cells was unchanged. Thus, strategies that supplement the stromal environment may enhance B lymphopoiesis in aged animals.

Vascular cell adhesion molecule 1-positive reticular cells express interleukin-7 and stem cell factor in the bone marrow.

In vitro studies have defined an essential role for stromal cells in supporting B-cell development, including production of lymphopoietic cytokines. It has been suggested that stromal cells are equivalent to adventitial reticular cells in the marrow; however, evidence of reticular cells producing cytokines has been difficult to obtain. Staining of bone marrow (BM) sections with antibodies to interleukin-7 (IL-7) showed a reticular pattern, mimicking that obtained using antibodies to vascular cell adhesion molecule 1 (VCAM-1), a molecule present on both stromal cells in vitro and reticular cells. To more closely examine cytokine production within normal marrow, an immunomagnetic separation scheme was devised to directly enrich VCAM-1+ stromal cells. Twenty to thirty percent of cells isolated in the VCAM-1+ fraction shared characteristics with stromal cells from long term BM cultures, including cellular morphology and expression of alkaline phosphatase and alpha actin. These were termed "reticular stromal" cells. Immunohistochemical staining showed that virtually all of the latter cells possessed cytoplasmic IL-7 protein, and about half expressed stem cell factor. In contrast with cultured stromal cells, very few had detectable macrophage-colony-stimulating factor. These data constitute the first report of cytokine expression by marrow reticular cells in vivo. The implications of this data with respect to the existence of stromal cell subsets and their regulation of lymphopoiesis is discussed.

Apoptosis and interleukin 7 gene expression in chronic B-lymphocytic leukemia cells.

mRNA for interleukin 7 (IL-7) was readily detected in leukemic cells immediately upon their removal from patients with chronic B-lymphocytic leukemia (B-CLL). IL-7 mRNA expression and IL-7 gene transcription were down regulated, however, when B-CLL cells were placed in culture at 37 degrees C for 4 hr. Down regulation of the IL-7 gene was prevented in cells maintained at 4 degrees C. Continued culture of B-CLL cells at 37 degrees C resulted in programmed cell death, or apoptosis, as evidenced by DNA fragmentation. The coincident kinetics of IL-7 gene down regulation and apoptosis suggested that IL-7 gene expression may be required for maintenance of CLL viability in vivo. Signals for IL-7 gene regulation and apoptosis induction were thus examined. Activation of normal B cells through their immunoglobulin receptors did not result in upregulation of IL-7 gene expression. Reagents required for CLL cell purification and culture also did not contribute to IL-7 gene regulation and apoptosis induction. IL-7 gene expression was retained and apoptosis was prevented, however, in CLL cells cultured on a monolayer of EA.hy926 human umbilical cord endothelial hybrid cells. Signals specifically presented by EA.hy926 cells supported both CLL cell viability and IL-7 gene expression, whereas culture of CLL cells on A549/8 carcinoma cells, the fusion partner used to generate the EA.hy926 cells, did not. Cell-cell contact was required, as culture supernatants did not prevent apoptosis. Specifically, IL-7 mRNA expression was retained and apoptosis was prevented only by contact with the endothelial cell hybrids. Preliminary data indicated that integrins expressed on CLL cells affected modulation of apoptosis and IL-7 gene regulation, suggesting that integrins may play significant roles in regulating viability of CLL cells.

Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates.

In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.

Cytokine production and heterogeneity of primary stromal cells that support B lymphopoiesis.

Many reports document that bone marrow stromal cells or their cytokine products can influence the formation of B cells in vitro. Most of this data comes from studies using lines or clones of stromal cells after multiple passage in culture, which could alter gene expression. Our aim in the present study was to determine which cytokines are produced by normal stromal cells under conditions that promote B lymphopoiesis. Primary cultured stromal cells were isolated on FACS from active Whitlock cultures. These cells proved to be relatively homogeneous in expression of cell surface antigens (CD44, VCAM-1, MECA10, and a molecule marked by hamster anti-mouse 8.28 monoclonal antibody). RNA from unselected Whitlock cultured adherent cells and sorted stromal cells from the same cultures were subjected to reverse transcriptase polymerase chain reaction to assess constitutive expression of several cytokine genes. Transcripts for interleukin-1 beta (IL-1 beta), IL-7, macrophage (M)-colony-stimulating factor (CSF), stem cell growth factor (SCGF), insulin-like growth factor 1 (IGF-1) and occasionally leukemia inhibitory factor were detected in RNA from intact cultures. Messages for IL-7, M-CSF, and SCGF were selectively contained within the isolated stromal cell fraction; whereas, IL-1 beta was found solely within the non-stromal cell fraction. IGF-1 was transcribed by both stromal cells and macrophages in Whitlock cultures. No evidence was found for constitutive expression of IL-1 alpha, IL-4, IL-6, or granulocyte-macrophage-CSF. This is in contrast to some reported stromal cell lines and clones. To determine if all primary stromal cells from active lymphopoietic cultures produced IL-7, the isolated cells were stained to reveal cytoplasmic IL-7 protein. A majority of the cells produced IL-7, but about 20% had no detectable IL-7 protein. Taken together, our results suggest that the primary stromal cells are a distinguishable cell type but functional subsets may exist. In regard to the differences in IL-7 production, the primary cell phenotype appears to mirror at least one division noted among the stromal cell lines.

Activity of stem cell factor and IL-7 in combination on normal bone marrow B lineage cells.

The production of B cells is regulated by soluble and cell contact signals presumably provided by bone marrow stromal cells. Among these is IL-7, a well characterized proliferative stimulus for a subset of pre-B cells. Stem cell factor (SCF), a stromal cell-derived cytokine with broad hemopoietic effects, has been reported to synergize with IL-7 to drive the proliferation and differentiation of B220- bone marrow cells into B220+ B cell precursors in long term culture. A subsequent report has cast doubt on this result by showing that SCF and IL-7 were incapable of producing mu+ pre-B cells after short term culture. Here, using the cell sorter to assure discrete separation of B220+ and B220- cells followed by soft agar culture to prevent interaction with accessory cells, we demonstrate that the combination of SCF and IL-7 does not stimulate the expansion or differentiation of B220- lymphoid precursors but can act synergistically in the clonal proliferation of B220+ cells.

Enrichment of primary lymphocyte-supporting stromal cells and characterization of associated B lymphocyte progenitors.

Studies of Whitlock/Witte long-term bone marrow cultures have revealed the necessity of two cell types for B lymphopoiesis, a stem cell and the stromal cell. While a number of stromal cell lines exist they have been found to be heterogeneous with respect to cell surface marker expression and growth factor production. Separation and analysis of fresh bone marrow stromal cells is, therefore, necessary to understand the regulation of lymphopoiesis in vivo. Here we report the early stages of such studies. We demonstrate that stromal cells, as assessed by morphology and alkaline phosphatase reactivity after short-term culture, are enriched in cellular aggregates that can be separated from bone marrow suspensions. Stromal cells are present in aggregates at a frequency of one per thousand cells, whereas marrow from which the aggregates have been removed contains only one stromal cell per fifty-thousand cells. These aggregates are able to form Whitlock cultures from greatly reduced numbers of initiating cells, indicating that they contain culturable B lineage precursors as well as stromal cells capable of supporting B lymphopoiesis. The aggregates appear to be naturally formed and provide a means to examine native B cell precursor-stromal cell contacts. We find little evidence for sequestering of late-stage B cell precursors within the aggregates. Terminal deoxynucleotidyl transferase-positive cells, on the other hand, are approximately three times more frequent in bone marrow aggregates, suggesting close contact between very early B cell progenitors and stromal cells within the aggregates. The finding that stromal cells are enriched in cellular aggregates is an important first step in the ultimate isolation of these cells from marrow suspensions, which is vital to understanding stromal cell function in vivo.

Further studies on growth factor production by the TC-1 stromal cell line: pre-B stimulating activity.

Adherent murine stromal cells support long-term in vitro lymphopoiesis or myelopoiesis dependent on the culture conditions used. A cell line, TC-1, isolated from long-term liquid murine marrow cultures under conditions approaching those permissive for lymphoid growth, has been found to produce an activity that acts synergistically with interleukin-3 (IL-3) or colony-stimulating factor-1 (CSF-1) to stimulate in vitro myeloid colonies, but which has no intrinsic colony-stimulating activity. We report here the presence of multiple growth factors in conditioned medium (CM) from the TC-1 line, including granulocyte-macrophage colony-stimulating factor (GM-CSF) (bioassay with antibody blocking and messenger RNA [mRNA] analysis), granulocyte CSF (G-CSF) and IL-4 (factor-dependent cell line bioassay), and CSF-1 (radioimmunoassay, mRNA) along with a pre-B cell inducing activity, which appears separate from these CSFs and segregates with the myeloid synergizing activity through anion exchange, sizing, and Conconavalin A chromatography. Because these activities are not yet purified to homogeneity, their identity or lack of identity remains an open question. Assays of TC-1 CM or cellular mRNA analysis have given negative results for IL-1, IL-2, IL-3, IL-6, and IL-7, and IL-6 does not stimulate pre-B cells in this assay. However, IL-4 and G-CSF do stimulate in vitro induction of pre-B cells from pre-B and B-cell-depleted Balb/C marrow and are present in CM by selective cell line assay. A monoclonal antibody to IL-4 that inhibited its pre-B inducing activity did not inhibit pre-B inducing activity of TC-1 CM. These data suggest the existence of a unique synergizing and pre-B inducing factor(s) in TC-1 CM. Given the known capacity of subliminal levels of growth factors to act synergistically, an alternate possibility is that these biologic phenomena represent the actions of low concentrations of growth factors acting synergistically and possibly associated with some core protein.

Natural killer cells in the thymus. Studies in mice with severe combined immune deficiency.

The relationship between NK cell and T cell progenitors was investigated by using mice with severe combined immune deficiency (scid). Scid mice are devoid of mature T and B cells because they cannot rearrange their Ig and TCR genes. However, they have normal splenic NK cells. Thymus of scid mice, although markedly hypocellular, contains cells that lyse YAC-1, an NK-sensitive tumor cell. By flow cytometry, two populations of cells were identified in the scid thymus. Eighty percent of the cells were Thy-1+, IL-2R(7D4)+, J11d+, CD3-, CD4-, CD8- whereas the remaining were IL-2R-, J11d-, CD3-, CD4-, and CD8-. By cell sorting, all NK activity was found in the latter population, which is phenotypically similar to splenic NK cells. To determine if the thymus contains a bipotential NK/T progenitor cell, J11d+, IL-2R+ cells were cultured and analyzed for the generation of NK cells in vitro. These cells were used because they resemble 15-day fetal and adult CD4- CD8- thymocytes that are capable of giving rise to mature T cells. Cultured J11d+ thymocytes acquired non-MHC-restricted cytotoxicity, but in contrast to mature NK cells, the resulting cells contained mRNA for the gamma, delta, and epsilon-chains of CD3. This suggests that J11d+ cells are early T cells that can acquire the ability to kill in a non-MHC-restricted manner, but which do not give rise to NK cells in vitro. The differentiative potential of scid thymocytes was also tested in vivo. Unlike bone marrow cells, scid thymocytes containing 80% J11d+ cells failed to give rise to NK cells when transferred into irradiated recipients. Together these results suggest that mature NK cells reside in the thymus of scid mice but are not derived from a common NK/T progenitor.

The xid mutation affects hemopoiesis in long term cultures of murine bone marrow.

Cells of the humoral immune system are particularly affected by a mutation at the X chromosome linked immunodeficiency disease (xid)locus. Although B cells are made in normal numbers, they fail to become phenotypically and functionally diverse. Consequently, poor antibody responses are mounted to certain types of Ag. There have been some indications that other types of hemopoietic cells may be influenced by the mutation and development of the humoral immune system is unusually dependent on the presence of T lymphocytes. We now describe an analysis of the lympho-hemopoietic environment studied with long term bone marrow cultures. Contrary to expectations, cultures initiated with cells from homozygous female or hemizygous male mice with the mutant allele established more quickly than normal. The accelerated initial growth pattern was clearly linked to the xid mutation. Artificial mixtures of marrow exhibited intermediate growth kinetics. Experiments with H-2 congenic and T6 chromosome marked cells did not reveal an intrinsic dominance of growth in nonadherent xid cells. Similar results were obtained with culture conditions which favored production of myeloid or lymphoid cells. These findings would be consistent with subtle changes in the bone marrow microenvironment resulting from the xid mutation. The pedigree of the mouse strains had a significant influence on lymphopoiesis in long term bone marrow cultures. Lymphocytes of BALB/c origin dominated over CBA/H background cells in cultures established from mixtures of the two, but this did not correlate with any functional deficiency in CBA/H stromal cells. In fact, establishment of an adherent layer was a rate-limiting step in initiating long term cultures and this could be achieved with a low dose inoculum of CBA/H marrow. Even more dramatic effects were found in hemopoietic cells from doubly defective C3H.nu/nu-xid mice. The bone marrow of these athymic animals contained normal numbers of granulocyte/macrophage progenitors. However, lymphoid cultures could not be reproducibly established with their cells and myelopoiesis was never observed in vitro. The relatively simple conditions which pertain in culture make it possible to appreciate effects of mutations and pedigree on hemopoiesis which are unremarkable in intact animals.

Lymphohemopoiesis in culture is prevented by interaction with adherent bone marrow cells from mutant viable motheaten mice.

Mice with the recessive "motheaten" (me) or "viable motheaten" (mev) mutations have severe immunologic disturbances and die at an early age. The function of hemopoietic progenitor cells and microenvironmental elements that regulate their growth and differentiation were studied in mev mice with two types of long term bone marrow cultures. Cells from bone marrow of homozygous defective mev/mev mice were non-productive under conditions that normally support replication of stem cells and production of neutrophil granulocytes. Similarly, in a different culture system, lymphocytes were produced from normal littermate, but not mev/mev bone marrow. Initial overgrowth of cells having macrophage-like characteristics occurred in both culture systems with marrow from defective mice. Co-cultures of normal and defective bone marrow cells were always non-productive. In contrast, supernatants of mev/mev bone marrow cultures did not have a detrimental effect on cultures of normal cells, implying that the suppression was cell-associated. Furthermore, there was no evidence for abnormal release of granulocyte or macrophage growth factors in mev bone marrow cultures. A small population of cells in mev/mev bone marrow cultures were morphologically similar to "stromal" cells that support lymphohemopoiesis. Certain culture strategies could be used to enrich for these. mev/mev stromal cells had affinity for normal lymphocytes; however, they did not support lymphocyte growth. The long term bone marrow cultures thus reveal an apparent imbalance in the regulatory mechanisms affected by these single gene mutations. This is manifested by preferential or aberrant growth of one type of adherent cell and a possible functional abnormality of stromal cells. mev mice could provide an ideal model for investigating cell-associated molecules that normally limit progenitor cell replication.

Transcriptional regulation of mu and delta gene expression in bone marrow pre-B and B lymphocytes.

Newly formed B cells first express IgM and subsequently display IgD on the cell surface. This is an ontologically, as well as developmentally, regulated process because IgD is virtually absent on neonatal splenic B cells. In the present studies we have examined, by means of nascent RNA chain labeling, the relative levels of mu to delta gene transcription in bone marrow B cells, pre-B cells, and earlier progenitors of B cells. Pre-B cells were obtained from Whitlock-type long term cultures of bone marrow cells from normal and C.B17 scid mice. Both populations were found to transcribe the delta gene at very low but detectable levels. A similarly low level of delta transcription was found to occur in surface IgM-positive cells from both cultured and freshly isolated bone marrow B cells. In all populations analyzed, termination of the majority of polymerases occurred within a discrete 1-kb region located between the microM and C delta I exons. Analysis of steady state RNA indicated that long term cultured bone marrow cells from normal mice produced both 2.7-kb normal sized microM mRNA as well as 2.9-kb aberrantly spliced I mu-mRNA, whereas those from C.B17 scid mice contained only aberrant sized mu-mRNA. In contrast to these results, our previous findings with spleen cells obtained from both neonatal and adult animals showed that delta gene transcription occurs at a relatively high level. Therefore, it is possible that activation of regulatory signals that allow polymerases to progress beyond the termination site 3' of the microM exons may occur when newly formed B cells migrate from the bone marrow to the splenic environment.

Relationships between B-lineage lymphocytes and stromal cells in long-term bone marrow cultures.

In long-term culture of mouse bone marrow, the growth and differentiation of B-lineage lymphocytes depends on interaction with adherent cells or their products. The objectives of these studies were to characterize the types of cells present in the supporting adherent layer as well as the physical relationships of these cells with lymphocytes. With an extensive panel of antibodies against hemopoietic and lymphocyte antigens, two discrete nonlymphoid populations were identified: macrophages and undefined, large cells which we termed "stromal cells". Lymphocyte clusters grew in actual contact with the latter cells only. Stromal cells lacked expression of most hemopoietic antigens, including the common leukocyte antigen, J11d, heat stable antigen (M1/69), Thy-1 and BP 1. Antigens expressed by stromal cells were detected by AA4.1, our 94.2 antibody, and antibody to the Forsmann antigen, but the most distinguishing characteristics of the lymphocyte-binding stromal cells were production of basement membrane components, laminin and collagen IV, and the extremely low uptake of acetylated low density lipoprotein (LDL). Using acetylated LDL uptake as a sorting criterion, the lymphocyte-binding stromal cells were separated from the macrophages, recultured and shown to support lymphocyte proliferation. We found the binding between stromal cells and lymphocytes to be highly selective and dependent on divalent cations; hence, specialized adhesion mechanisms may have a role in B cell development. Moreover, our studies suggest that phosphatidylinositol-anchored cell surface molecules may be involved in this adhesion. Our findings demonstrate the possibility that a single cell type provides physical support and proliferation stimuli for early B-lineage cells. This accessory cell is not a macrophage; rather, it has features of an endothelial or epithelial cell.

Lack of dendritic Thy-1+ epidermal cells in mice with severe combined immunodeficiency disease.

C.B-17 scid (severe combined immunodeficiency disease) mice were used to evaluate the relationship of dendritic Thy-1+ epidermal cells (EC) to T lymphocytes (deficient in scid) and to NK cells (replete in scid). Epidermis from scid mice was deficient in dendritic Thy-1+ cells as determined by immunofluorescent staining of epidermal whole mounts. Similarly, epidermal cell suspensions from scid mice failed to proliferate in response to Con A, as compared with epidermal cell suspensions from C.B-17 control mice. Transplantation of normal bone marrow into scid mice reconstituted morphologically identifiable dendritic Thy-1+ EC in whole mounts, as well as Con A responsiveness of EC suspensions, thus indicating that the deficiency in dendritic Thy-1+ EC in scid mice is at the precursor level. These studies demonstrate that Thy-1+ EC are more closely related to T lymphocytes than to NK cells.