Limitin: An interferon-like cytokine that preferentially influences B-lymphocyte precursors.

We have identified an interferon-like cytokine, limitin, on the basis of its ability to arrest the growth of or kill lympho-hematopoietic cells. Limitin strongly inhibited B lymphopoiesis in vitro and in vivo but had little influence on either myelopoiesis or erythropoiesis. Because limitin uses the interferon alpha/beta receptors and induces interferon regulatory factor-1, it may represent a previously unknown type I interferon prototype. However, preferential B-lineage growth inhibition and activation of Janus kinase 2 in a myelomonocytic leukemia line have not been described for previously known interferons.

Suppression of B lymphopoiesis during normal pregnancy.

We describe a dramatic reduction in numbers and activity of committed B lymphocyte precursors in the bone marrow of normal pregnant mice. Changes in cells responsive to IL-7 were evident as early as 6.5 d of pregnancy and values were < 10% of normal at parturition. B lineage precursors, identified by display of CD45R and absence of surface IgM, were also substantially depressed, and subpopulations representing different stages in the B lineage were assessed by three-color flow cytometry. Early pro-B cells are medium to large in size and have been previously characterized by low expression of the heat-stable antigen (HSA). This category of cells was not reduced, and in fact may have been slightly elevated, during pregnancy. In contrast, all subsequent populations of B lineage precursors, defined by patterns of expression of heat-stable and CD43 antigens, were substantially depressed. The immediate precursors of B cells (small pre-B cells) were identified by small size, expression of CD45R, absence of CD43, and lack of surface IgM. These were the most reduced of any phenotypically defined population in bone marrow. Numbers of newly formed B cells, characterized by the presence of sIgM, but not sIgD, were also diminished. However, B cells with a mature phenotype (sIgM+, sIgD+) were present in normal to somewhat elevated numbers. Mitogen-responsive B cells clonable in a semisolid agar assay were not significantly affected. A bromodeoxyuridine (BrdU) labeling technique was used to evaluate mitotic activity, which revealed an increased proportion of long-lived lymphocytes in the bone marrow of pregnant mice. These observations indicate that B lymphopoiesis is markedly downregulated during pregnancy and that all precursor populations beyond the early pro-B cell stage are affected. The pregnancy-related changes in bone marrow were selective for B lineage precursors, as cells expressing myeloid and erythroid markers were not reduced. In spleen, evidence was obtained for partial depletion of one subset of B cells. These cells, which have been reported to be recent immigrants from marrow, are characterized as having high levels of sIgM and HSA. Changes in other major B lymphocyte subsets in the spleen were less remarkable. When considered with results from the BrdU labeling procedure, the findings indicate that both production and export of lymphocytes from marrow may be substantially decreased. Numbers of B cell precursors were higher in postpartum animals whose litters were removed at birth, suggesting that lactation may prolong regeneration of lymphocyte production.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long-term bone marrow cultures.

A new panel of mAbs was prepared to a stromal cell line known to support lymphocytes in Whitlock-Witte type long-term bone marrow cultures. These antibodies were then screened with a cell adhesion assay and four were selected that inhibited the binding of B lineage cells to stromal cell monolayers. Immunofluorescent and biochemical analyses revealed that these new antibodies detected epitopes of the previously described Pgp-1/CD44 antigen complex. Addition of Pgp-1/CD44 antibodies to Dexter-type long-term bone marrow cultures completely prevented emergence of myeloid cells and they also blocked lymphocyte growth in Whitlock-Witte type cultures. mAbs MEL-14, LFA-1, and CD45R did not inhibit under the same conditions and there was no apparent relationship to Ig isotype. Adherent layers in treated cultures were not unusual in terms of morphology and the antibodies did not affect factor-dependent replication of lymphoid or myeloid progenitor cells. Therefore, the mechanism of inhibition may not involve direct toxicity to precursors or microenvironmental elements. Previous studies in humans and mice have implicated Pgp-1/CD44-related glycoproteins in the migration of peripheral lymphoid cells, as well as interactions of cells with the extracellular matrix. These findings suggest that they may also be critical for formation of lymphoid and myeloid cells within bone marrow.

Gene regulatory networks orchestrating B cell fate specification, commitment, and differentiation.

The B cell developmental pathway represents a leading system for the analysis of regulatory circuits that orchestrate cell fate specification, commitment, and differentiation. We review the progress that has been achieved in the identification and characterization of regulatory components of such circuits, including transcription factors, chromatin modifying proteins, and signaling molecules. A comprehensive developmental model is proposed that invokes sequentially acting regulatory networks which dictate the generation of B cells from multipotential hematopoietic progenitors.

The pharmacologic and expectancy effects of alcohol on social anxiety in individuals with social phobia.

Individuals with social phobia are at an increased risk for alcohol problems. Individuals with social phobia could increase their risk for pathological alcohol use if they drink as a means of coping with anxiety-provoking social situations. Providing a circumscribed test of this view, we evaluate the effect of alcohol on the intensity of social phobia anxiety responses. Sixty-one individuals with social phobia gave two speech challenges in front of a group ('social anxiety challenge'), one occurring before and one after they consumed either: (a) an alcoholic drink they were told contained alcohol ('alcohol group'), (b) a non-alcoholic drink they were told contained alcohol ('placebo group'), or, (c) a non-alcoholic drink they were told contained no alcohol ('control group'). Both the alcohol group and the placebo group showed greater reduction in performance anxiety from the first to the second speech challenge than did the control group. Further, there was a strong trend in the data for the alcohol group to show greater reduction in performance anxiety from the first to the second speech challenge than did the placebo group. We concluded from these findings that the pharmacologic effects of alcohol and the belief that one consumed alcohol decrease social performance anxiety in an additive fashion. These results provide direct support for the negatively reinforcing properties of alcohol and are consistent with the view that symptom reduction may motivate alcohol use among socially phobic individuals.

Recognition of murine integrin beta 1 by a rat anti-stromal cell monoclonal antibody.

Previous studies with the rat monoclonal antibody KMI6 had localized its antigen in vivo to a discrete subpopulation of marrow stromal cells. The KMI6 antigen has now been identified as the murine homolog of integrin beta 1 by amino acid sequence analysis and by cross-reactivity with antiserum to the avian integrin beta 1. The relative tissue abundance of murine integrin beta 1 was determined by Western blot. Although immunoperoxidase staining of fixed murine hematopoietic tissues demonstrated an abundance of intracellular beta 1, few primary-derived cells of lymphohematopoietic origin were surface positive as assessed by immunofluorescence and flow cytometry. Fetal erythroblasts provided the only exception. In contrast, the antigen was readily detected on the surface of several cultured cell lines in association with a variety of alpha chains. The biochemical properties of the surface labeled murine integrin beta 1 were similar to those of its human counterpart, exhibiting an altered electrophoretic migration under reduced conditions or following N-glycanase treatment. The antibody recognition of the protein was insensitive to glycosylation state, presence of divalent cations, detergents, or transfer to a nitrocellulose membrane. However, on Western blot, the epitope was lost on reduction of the protein, suggesting that it is conformation dependent. These data indicate that although KMI6 epitope is widely distributed, its surface expression in vivo may be restricted within lymphohemopoietic tissues.

Pregnancy-related steroids are potential negative regulators of B lymphopoiesis.

B lymphopoiesis is selectively suppressed in normal pregnant mice, suggesting that fluctuations in systemic hormone levels might influence local events within bone marrow. This has now been tested by sustained experimental elevation of sex steroids by hormone-containing pellet implants. We found that while numbers of total nucleated cells declined after treatment with estrone, beta-estradiol, or estriol, there was preferential suppression of B-lymphocyte lineage precursors. Progesterone pellets had no effect when used alone, but mice exposed to progesterone were sensitive to several-logarithm lower concentrations of estrogen. Changes in subpopulations of B-lymphocyte lineage cells with hormone pellets were similar to those previously recorded in pregnancy. B-lymphocyte lineage precursors in male and female mice were sensitive to these sex hormones. Acute treatment with single injections of water-soluble beta-estradiol allowed temporal effects on B-lineage cells to be documented. With this protocol, total numbers of nucleated cells and myeloid progenitor cells remained unchanged. Interleukin 7-responsive precursors dramatically declined within 1 day of injection, suggesting that estrogen influences that stage in the B-lymphocyte lineage. There was a subsequent sharp drop in small pre-B cells 4 days after this transient elevation in estrogen. These experiments demonstrate that B lymphopoiesis is sensitive to negative regulation by sex steroids. They extend findings made with pregnant animals and parallel previous studies of the thymus. Sex steroids might contribute to control of steady-state lymphopoiesis, and fluctuations in their levels could have implications for human disease.

Sex hormones as negative regulators of lymphopoiesis.

B lymphocytes, together with cells of seven other lineages, are made in large numbers from precursors in the bone marrow. Using cell culture models and recombinant proteins, progress has been rapid in identifying cytokines which could potentially regulate the proliferation, differentiation and migration of B-cell precursors. However, we still know little about molecular mechanisms which are important for maintaining steady-state conditions in vivo. B lymphopoiesis is severely diminished during pregnancy in normal mice and this provided a clue that sex hormones might be important negative regulators. Administration of estrogens alone, or in combination with progesterone, preferentially suppressed IL-7 responding cells and their progeny in bone marrow. There is precedent for these observations in the thymus, which transiently involutes during pregnancy, and also atrophies following estrogen treatment. The actual mechanism(s) through which sex steroids influence lymphopoiesis remain unclear, but cell culture experiments should be informative about potential interactions between hormones, the bone marrow microenvironment, and lymphocyte precursors. These findings raise a number of other important issues. For example, we need to learn if sex steroids are produced and/or concentrated locally within the marrow, if human lymphopoiesis is sensitive to these hormones, and if production of lymphocytes can be augmented in aging and in immunodeficiency by hormone manipulation.

Estrogen influences the differentiation, proliferation, and survival of early B-lineage precursors.

B lymphocyte production in murine bone marrow is negatively regulated by sex steroids and the aim of this study was to identify early hormone sensitive checkpoints. Estrogen (E2) treatment reduced cmu(+) pre-B cells, a change that occurred concomitantly with decreased Ig gene rearrangements and rag-1 transcripts. Estrogen decreased B lineage precursors in Ig transgenic mice, demonstrating that hormonal regulation is independent of the recombination process. B lineage precursors in Bcl-2 transgenic mice were resistant to estrogen treatment, suggesting that life/death decisions are involved in hormonal regulation. A previously uncharacterized population of CD43(-)cmu(-) B lineage precursors was identified in normal, Ig transgenic, and RAG(-/-) mice after estrogen treatment, revealing that down-regulation of CD43 can occur independent of Ig heavy chain expression. These cells expressed transcripts for both tdt and bcl-2, characteristics of early B-cell precursors. BrdU incorporation analysis revealed that the mitotic activity of early B-lineage cells is reduced in hormone-treated mice. We conclude that sex steroids modulate the production of B-lineage cells by influencing the differentiation, proliferation, and survival of early B-cell precursors. These findings are informative about mechanisms of hormonal regulation, as well as the significance of some differentiation-related events. (Blood. 2000;95:2059-2067)

Loss of c-kit accompanies B-lineage commitment and acquisition of CD45R by most murine B-lymphocyte precursors.

Using surface markers, we identified two bone marrow (BM) subsets enriched for TdT+ cells on the brink of CD45R acquisition. These two populations, Lin-c-kitLo and Lin-c-kit-, consisting of 35.4% and 7. 4%, respectively, TdT+ cells, generated B-lineage cells in overnight cultures. Approximately half of the c-kitLo B-lineage precursors were bipotential, yielding myeloid and lymphoid progeny, whereas most that were c-kit- gave rise only to lymphocytes. Analysis of B-lineage progression during a finite culture period showed that the most mature precursors were concentrated in the Lin-c-kit- population. Moreover, a majority of the earliest CD45R+ pro-B cells in BM, identified as CD45R+ CD43(+) BP-1(-) CD25(-) natural killer (NK)1.1(-) sIgM-, were also c-kit-. These c-kit- cells, like their c-kitLo counterparts, expressed TdT, proliferated in response to interleukin (IL)-7, and generated sIgM+ cells. These data suggest that TdT expression is initiated as c-kit downregulation begins in Lin- cells, with progressive loss of c-kit during B-lineage differentiation. CD45R expression is initiated during the transition from c-kitLo to c-kit- with many cells losing c-kit before acquiring CD45R. The ability to isolate highly enriched populations of viable CD45R- precursors will be instrumental in characterizing the earliest B-lineage cells.

Pregnancy: a clue to normal regulation of B lymphopoiesis.

The large-scale production of lymphocytes in the bone marrow reflects a delicate balance between positive and negative regulatory signals. For instance, interleukin 7 (IL-7) provides a positive signal, and appears to be both essential and limiting in the mouse. However, much less is understood concerning the negative molecular signals that may limit the output of lymphocytes. Here, Paul Kincade and colleagues discuss how a chance observation with pregnant mice revealed that sex steroids can act as negative regulators of B lymphopoiesis, and may do so under normal steady-state conditions.

Characteristics of early murine B-lymphocyte precursors and their direct sensitivity to negative regulators.

Recently, a collection of surface markers was exploited to isolate viable Lin(-) TdT(+) cells from murine bone marrow. These early pro-B cells were enriched for B-lineage lymphocyte precursor activity measured by short-term culture and had little responsiveness to myeloid growth factors. Early precursors can be propagated with remarkably high cloning frequencies in stromal cell-free, serum-free cultures, permitting this analysis of direct regulatory factors. Expression of the interleukin-7 receptor (IL-7Ralpha) chain marks functional precursors and IL-7 is necessary for progression beyond the CD45RA(+) CD19(-) stage. Efficient survival and differentiation were only observed when stem cell factor and Flt-3 ligand were also present. IL-7-responsive CD19(+) precursors are estrogen resistant. However, B-lineage differentiation was selectively abrogated when highly purified Lin(-) precursors were treated with hormone in the absence of stromal cells. In addition, early stages of B lymphopoiesis were arrested by limitin, a new interferon (IFN)-like cytokine as well as IFN-alpha, IFN-gamma, or transforming growth factor beta (TGF-beta), but not by epidermal growth factor (EGF). Lin(-) TdT(+) early pro-B cells are shown here to be CD27(+) AA4.1(+/-)Ki-67(+) Ly-6C(-) Ly-6A/Sca-1(Lo/-)Thy-1(-)CD43(+) CD4(+/-)CD16/32(Lo/-)CD44(Hi) and similar in some respects to the "common lymphoid progenitors" (CLP) identified by others. Although early pro-B cells have lost myeloid differentiation potential, transplantation experiments described here reveal that at least some can generate T lymphocytes. Of particular importance is the demonstration that a pivotal early stage of lymphopoiesis is directly sensitive to negative regulation by hormones and cytokines.

Disagreement about the occurrence of male-to-female intimate partner violence: a qualitative study.

This work explored the reasons underlying inter-partner disagreement about the occurrence of intimate partner violence (IPV). Research indicates that partners often do not agree about episodes of conflict in their relationship. We conducted interviews with 48 women and men with and without histories of IPV to investigate this lack of agreement. Participant responses were analyzed and themes were identified about why men and women disagree about episodes of conflict. The main results indicate that participants think women and men remember differently; women remember more than men, both choose what they want to remember, and both remember that they were right in the conflict. This work contributes to understanding the disagreement that occurs between partners. Many of these findings have never been suggested by other IPV researchers. The broad-reaching implications of this study include improvement in the accuracy of measuring IPV.

Identification of very early lymphoid precursors in bone marrow and their regulation by estrogen.

Estrogen is a negative regulator of lymphopoiesis and provides an experimental tool for probing relationships between lymphocyte precursors and stem cells. We found that expression of lymphocyte-associated genes and immunoglobulin (Ig) gene rearrangement occurred before CD45R acquisition. Lymphoid-restricted progenitors that were Lin(-)IL-7R alpha(+)c-kit(lo)TdT(+) (lineage marker(-), interleukin receptor 7 alpha(+), c-kit(lo) and terminal deoxynucleotidyl transferase(+)) were selectively depleted in estrogen-treated mice; within a less differentiated Lin-c-kit(hi) fraction, functional precursors of B and T, but not myeloid, cells were also selectively depleted. TdT and an Ig heavy chain transgene were detected within a hormone-regulated Lin(-)c-kit(hi)Sca-1(+)CD27(+)Flk-2(+)IL-7R alpha(-) subset of this multipotential progenitor population. Identification of these extremely early lymphoid precursors should facilitate investigation of the molecular mechanisms that control lineage-fate decisions in hematopoiesis.

Expression of bright at two distinct stages of B lymphocyte development.

The B cell regulator of Ig heavy chain transcription (Bright) is a DNA-binding protein that was originally discovered in a mature Ag-specific B cell line after stimulation with IL-5 and Ag. It binds to the intronic heavy chain enhancer and 5' of the V1 S107 family V(H) promoter. Several studies suggested that Bright may increase transcription of the heavy chain locus, and expression in cell lines was limited to those representing mature B cells. We have now analyzed normal hemopoietic tissues for the expression of Bright during B lymphocyte differentiation. We expected to find Bright expression in a subset of mature spleen cells, but also observed Bright in a subset of normal B lymphocytic progenitors in both adult bone marrow (BM) and in fetal liver as early as day 12 of gestation. Bright was also expressed in the small percentage of CD4(low) cells in the thymus that are newly arrived from the BM and are not yet committed to the T lymphocyte lineage, but was not observed at later stages of T cell differentiation in either the spleen or thymus. Bright mRNA was not detected in the immature B lymphocytes that initially populate the spleen after migration from the BM. In addition, new splice variants of Bright were observed in fetal tissues. Thus, Bright expression is highly regulated in normal murine lymphocytes and occurs both early and late during B cell differentiation. These findings may have important implications for the function of Bright in regulating Ig transcription.

Early B-lymphocyte precursors and their regulation by sex steroids.

This review describes an improved characterization of early B-lymphocyte precursors in mice and the remarkable sensitivity of the same cells to hormones. The nuclear enzyme terminal deoxynucleotidyl transferase (TdT) was used as a marker to image and characterize bone marrow cells lacking all lineage-associated markers. Most early TdT+ precursors have a distinctive density of c-kit and express the interleukin-7Ralpha chain, as well as flt-3/flk2, but lack CD34. An understanding of those cell surface properties made it possible to obtain highly enriched, viable cells with the potential to give rise to CD19+ lymphocytes in culture. A series of other flow cytometry and culture experiments suggested a possible differentiation sequence for these early pro-B cells. This new model was used to advantage in our studies of sex steroids. It appears that early precursors represent a hormone-sensitive control point for determining numbers of new B lymphocytes that are produced within bone marrow. We also compare and contrast these findings with B lymphopoiesis in humans.

Relatively normal human lymphopoiesis but rapid turnover of newly formed B cells in transplanted nonobese diabetic/SCID mice.

Human B lineage lymphocyte precursors in chimeric nonobese diabetic/SCID mice transplanted with umbilical cord blood cells were directly compared with those present in normal bone marrow. All precursor subsets were represented and in nearly normal proportions. Cell cycle activity and population dynamics were investigated by staining for the Ki-67 nuclear Ag as well as by incorporation experiments using 5-bromo-2'-deoxyuridine. Again, this revealed that human B lymphopoiesis in chimeras parallels that in normal marrow with respect to replication and progression through the lineage. Moreover, sequencing of Ig gene rearrangement products showed that a diverse repertoire of V(H) genes was utilized by the newly formed lymphocytes but there was no evidence for somatic hypermutation. The newly formed B cells frequently acquired the CD5 Ag and had a short life span in the periphery. Thus, all molecular requirements for normal B lymphocyte formation are present in nonobese diabetic/SCID mice, but additional factors are needed for recruitment of B cells into a fully mature, long-lived pool. The model can now be exploited to learn about species restricted and conserved environmental cues for human B lymphocyte production.