B cell deficiency progresses with lineage maturation in nude.X-linked immunodeficient mice B cell deficiency progresses with lineage maturation.

Previously we showed that unlike normal, nude, or X-linked immune deficient (xid) mice, nude.xid mice are deficient in bone marrow pre-B cell targets for Abelson murine leukemia virus transformation. We show that nude.xid bone marrow is deficient in both CD45(B220)+ and CD45(B220)- surface (s)IgM- progenitors that give rise to B cell colonies in Whitlock-Witte cultures. CD45(B220)+ precursors had normal differentiation potential in vitro. CD45(B220)- precursors differentiated into CD45(B220)+ cells at the same rate as normal controls, but acquired sIgM at a much slower rate. These results correlated with the observation that in nude.xid mice the severity of B lineage defects correlates with maturity: a profound (ninefold) deficit of sIgM+, CD45(B220)+ mature B cells, a fivefold deficit in the sIgM-, CD45(B220)+ precursors of short term B cell colonies (colonies forming within 4-5 days in Whitlock-Witte cultures), and a moderate (twofold) decrease in the frequency of sIgM-, CD45(B220)- (less mature) precursors of long term B cell colonies (colonies forming after 14 days of Whitlock-Witte culture. Thus the combination of the nude and xid mutations produces a deficiency in early B cell progenitors and the deficiency becomes more profound with further maturation. Therefore the lack of mature B cells is the result of a cascade effect. Inasmuch as bone marrow progenitors are affected, and these are the source of the vast majority of B cells, most B cells are affected by the xid mutation and the xid defect cannot be attributed to a loss of a fetal lineage of B cells. These results suggest that xid affected cells lack the capacity to progress efficiently through differentiation in the absence of an exogenous factor(s) that is dependent on the product of a normal allele at the nude locus. This product might be supplied in vivo by a T cell or T cell-dependent source and/or epithelial elements such as bone marrow stromal cells all of which are known to be affected by the nude mutation.     

A cell surface glycoprotein virus inhibitor that is not interferon

The possible relationship between a newly isolated glycoprotein virus inhibitor and interferon was assessed. Comparisons of the cell surface glycopeptide, obtained from mouse cerebral cortex, and interferon included antiviral activity, radioimmune assays, and the ability of antibodies raised against the brain cell surface glycoprotein (BCSG) and against mouse L cell interferon to precipitate the biological activity. BCSG was able to inhibit virus replication but only in a transient fashion. Although anti-BCSG precipitated a major portion of the radiolabelled inhibitor in a double antibody assay, anti-mouse interferon did not. Over 90% of the inhibitory activity was removed with anti-BCSG and $\text{Staphylococcus}$ protein A while anti-mouse interferon removed little, or none, of the activity under similar reaction conditions. Other properties of the BCSG that distinguish it from interferon are presented.     

Targeted disruption of ephrin B1 in cells of myeloid lineage increases osteoclast differentiation and bone resorption in mice

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.     

A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice.

Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a good model system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest cell lineage, an efficient method has been devised for manipulating the mouse genome through the Cre-loxP system. We generated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNA recombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg line carries a lacZ gene downstream of a chicken beta-actin promoter and a "stuffer" fragment flanked by two loxP sequences, so that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre lines crossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation in mammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis, purification, and genetic manipulation of the mammalian neural crest cells.     

A cell surface protein that binds avian hepatitis B virus particles.

We have identified a 180-kDa cellular glycoprotein (gp180) that binds with high affinity to duck hepatitis B virus (DHBV) particles. The protein was detected by coprecipitating labeled duck hepatocyte proteins with virions or recombinant DHBV envelope proteins, using nonneutralizing monoclonal antibodies to the virion envelope. Binding of gp180 requires only the pre-S region of the viral large envelope protein, since recombinant fusion proteins bearing only this region efficiently coprecipitate gp180. The DHBV-gp180 interaction is blocked by two independent neutralizing monoclonal antibodies. The protein is found on both internal and surface membranes of the cell, and the species distribution of gp180 binding activity mirrors the known host range of DHBV infection. Functional gp180 is expressed in a wide variety of tissues in susceptible ducks.     

Antinuclear antigen B cells that down-regulate surface B cell receptor during development to mature, follicular phenotype do not display features of anergy in vitro

We previously demonstrated that B cells expressing a transgenic BCR with "dual reactivity" for the hapten arsonate and nuclear autoantigens efficiently complete development to follicular phenotype and stably reside in follicles in vivo. These B cells express very low levels of surface IgM and IgD, suggesting that they avoid central deletion and peripheral anergy by reducing their avidity for autoantigen via surface BCR (sBCR) down-regulation. Since a variety of states of B cell anergy have been previously described, a thorough examination of the functional capabilities of these B cells was required to test this hypothesis. In this study, we show that surface Ig cross-linking induces amounts of proximal BCR signaling in these B cells commensurate with their reduced sBCR levels. Functionally, however, they are comparable to nonautoreactive B cells in cell cycle progression, up-regulation of activation and costimulatory molecules, and Ab-forming cell differentiation when treated with a variety of stimuli in vitro. In addition, these B cells can efficiently process and present Ag and are capable of undergoing cognate interaction with naive TCR-transgenic T cells, resulting in robust IL-2 production. Together, these data reveal a lack of intrinsic anergy involving any known mechanism, supporting the idea that this type of antinuclear Ag B cell becomes indifferent to cognate autoantigen by down-regulating sBCR.     

Expression of human complement receptor type 2 (CD21) in mice during early B cell development results in a reduction in mature B cells and hypogammaglobulinemia

Complement receptor (CR) type 2 (CR2/CD21) is normally expressed only during the immature and mature stages of B cell development. In association with CD19, CR2 plays an important role in enhancing mature B cell responses to foreign Ag. We used a murine Vlambda2 promoter/Vlambda2-4 enhancer minigene to develop transgenic mice that initiate expression of human CR2 (hCR2) during the CD43(+)CD25(-) late pro-B cell stage of development. We found peripheral blood B cell numbers reduced by 60% in mice expressing high levels of hCR2 and by 15% in mice with intermediate receptor expression. Splenic B cell populations were altered with an expansion of marginal zone cells, and basal serum IgG levels as well as T-dependent immune responses were also significantly decreased in transgenic mice. Mice expressing the highest levels of hCR2 demonstrated in the bone marrow a slight increase in B220(int)CD43(+)CD25(-) B cells in association with a substantial decrease in immature and mature B cells, indicative of a developmental block in the pro-B cell stage. These data demonstrate that stage-specific expression of CR2 is necessary for normal B cell development, as premature receptor expression substantially alters this process. Alterations in B cell development are most likely due to engagement of pre-B cell receptor-mediated or other regulatory pathways by hCR2 in a CD19- and possibly C3 ligand-dependent manner.     

A conserved chromatin architecture marks and maintains the restricted germ cell lineage in worms and flies

In C. elegans, mRNA production is initially repressed in the embryonic germline by a protein unique to C. elegans germ cells, PIE-1. PIE-1 is degraded upon the birth of the germ cell precursors, Z2 and Z3. We have identified a chromatin-based mechanism that succeeds PIE-1 repression in these cells. A subset of nucleosomal histone modifications, methylated lysine 4 on histone H3 (H3meK4) and acetylated lysine 8 on histone H4 (H4acetylK8), are globally lost and the DNA appears more condensed. This coincides with PIE-1 degradation and requires that germline identity is not disrupted. Drosophila pole cell chromatin also lacks H3meK4, indicating that a unique chromatin architecture is a conserved feature of embryonic germ cells. Regulation of the germline-specific chromatin architecture requires functional nanos activity in both organisms. These results indicate that genome-wide repression via a nanos-regulated, germ cell-specific chromatin organization is a conserved feature of germline maintenance during embryogenesis.     

Age-dependent changes in B lymphocyte lineage cell populations of autoimmune-prone BXSB mice

BXSB mice, a recently developed autoimmune strain, develop a human lupus-like disease with B cell hyperplasia in peripheral lymphoid organs. Unlike other experimental models of autoimmunity and human lupus, BXSB male mice manifest accelerated autoimmune phenomena through the influence of a Y chromosome-linked enhancing factor. The present studies were performed to investigate the features of B lymphopoiesis in BXSB mice and to determine whether differences exist between BXSB males and females in this respect. B lineage cell populations in the marrow of BXSB mice were identified phenotypically by studying the cytoplasmic mu-heavy chains of IgM (c mu), and functionally by their ability to acquire clonability and sIg in short-term liquid cultures. Male BXSB mice became deficient in both the precursors of functional B cells and c mu + pre-B cells by the age of 8 to 12 wk. This followed a transient increase in this population, which peaked when the mice were 2 to 4 wk old. In females, substantial numbers of functional B cell precursors and c mu + cells were maintained until more than 4 mo of age. Cells lacking Ig but bearing a B lineage cell antigen (14.8) were elevated in numbers in both BXSB males and females until 16 wk of age when compared to normal strains of mice. At the time pre-B cells and functional B precursors were elevated in numbers, some sIg- cells were shown to form colonies in mitogen-stimulated semisolid agar cultures without a period of preculture. Most of these sIg- cells seemed to bear the B lineage cell antigen (14.8). They were independent of both G-10 adherent regulatory cells and Thy-1+ cells for their colony formation. These results indicate that B lymphocyte formation may be maintained in a hyperactive state in BXSB females, whereas males become deficient in B cell precursors very early in life. This early decline might be related to the accelerated development of autoimmune disease in BXSB mice. Bone marrow transplantation studies showed that these unusual characteristics of B lymphopoiesis were reciprocally transferable with unseparated bone marrow cells between BXSB males and females. This finding indicates that sex hormones are not a critical variable in abnormal B lymphocyte formation in this strain, and that the premature deficiency of immediate B precursors in males may be regulated by a genetic factor(s) located on the Y chromosome.     

Changes in B lineage cell population in liver and spleen of normal neonatal mice

The frequency and characteristics of B lymphocyte lineage cells in neonatal murine liver and spleen were studied during the first 10 days after birth. These were distinguished as B cells with surface IgM (slgM), immediate precursors of B cells (pre-B cells) lacking slgM but containing micron-heavy chains of IgM, and earlier precursors that did not synthesize immunoglobulin but could be detected with monoclonal 14.8 antibody. Experiments were also done to relate these to cells capable of clonal proliferation in mitogen-containing semisolid agar cultures and cells that acquire this function only after preculture in liquid medium. Newborn liver contained large numbers of early precursors as well as pre-B cells, and culture studies revealed that a majority of the colony-forming B cells present at that time were slg-. Adherent accessory cells in newborn liver suspensions facilitated the maturation of these into functional B cells in vitro. At most ages, however, numbers of slg+ B cells detected in that tissue were surprisingly low. Possible explanations for this include a rapid exit of newly formed B cells and their immediate precursors from liver and/or a high rate of abortive lg gene rearrangements during the neonatal period. In contrast, whereas the spleen contained early precursors and pre-B cells at birth, these cells steadily declined in number with age as the numbers of slgM+ B cells increased. Adherent cells in liver but not spleen of immunodeficient CBA/N mice suppressed B lymphocyte formation in semisolid or liquid cultures. These observations document population dynamics in B lineage cells during a critical period of development.     

Antibody to a novel 95-kDa surface glycoprotein on human B cells induces calcium mobilization and B cell activation

These findings characterize a 95-kDa glycoprotein on the surface of B lymphocytes recognized by the mAb G28-8. This protein (designated Bgp95), previously classified as a CD39 molecule, is unique based on functional, cell distribution, and immunochemical criteria. Biochemical analyses revealed that Bgp95 is a 95-kDa glycoprotein with N-linked carbohydrate and is reduced to about 70-kDa after treatment with endoglycopeptidase F. In functional studies, stimulation by G28-8 mAb or its F(ab')2 fragments induced a G0 to G1 cell cycle transition and was synergistic with PMA, anti-mu, or anti-CDw40 in stimulating proliferation of resting B cells. G28-8 mAb also could induce increases of cytoplasmic free calcium concentration [Ca2+]i in a subpopulation of tonsillar or peripheral blood B cells. The G28-8 mAb alone induced a steady increase in [Ca2+]i detectable even 1 h after stimulation. Cross-linking the G28-8 mAb with a second mAb specific for murine kappa light chains induced a more rapid increase of [Ca2+]i which peaked at 10 to 20 min and then declined. At 1 h after stimulation, [Ca2+]i was higher in B cells stimulated with G28-8 alone than in B cells stimulated with G28-8 plus anti-kappa. The same conditions of cross-linking with the anti-kappa which increased the kinetics of the [Ca2+]i response decreased the proliferative response which otherwise followed co-incubation of the mAb with B cell growth factor or PMA. Thus, conditions leading to rapid but transient [Ca2+]i increase via Bgp95 may not be as effective at stimulating B cell proliferation as conditions favoring a slower prolonged [Ca2+]i response. Although the Bgp95 molecule is present on activated buoyant tonsillar B cells, mAb to Bgp95 did not trigger [Ca2+]i increases in these cells. These results suggest that the Bgp95 protein may function in early B cell activation and that its signal mechanisms are altered by the activation state of the cell.     

Transgenic expression of Helios in B lineage cells alters B cell properties and promotes lymphomagenesis

Helios, a member of the Ikaros family of DNA-binding proteins, is expressed in multipotential lymphoid progenitors and throughout the T lineage. However, in most B lineage cells, Helios is not expressed, suggesting that its absence may be critical for B cell development and function. To test this possibility, transgenic mice were generated that express Helios under the control of an Ig mu enhancer. Commitment to the B cell lineage was unaltered in Helios transgenic mice, and numbers of surface IgM(+) B cells were normal in the bone marrow and spleen. However, both bone marrow and splenic B cells exhibited prolonged survival and enhanced proliferation. B cells in Helios transgenic mice were also hyperresponsive to Ag stimulation. These alterations were observed even though the concentration of ectopic Helios in B lineage cells, like that of endogenous Helios in thymocytes, was well below the concentration of Ikaros. Further evidence that ectopic Helios expression contributes to B cell abnormalities was provided by the observation that Helios transgenic mice developed metastatic lymphoma as they aged. Taken together, these results demonstrate that silencing of Helios is critical for normal B cell function.     

Regulation of cell division of mature B cells by ionomycin and phorbol ester.

The growth of a human B lymphoma cell line B104, an experimental model for mature B cells, was inhibited by ionomycin but not 12-O-tetradecanoylphorbol-13-acetate (TPA). Ionomycin inhibited B104 cells from entering into the M phase of the cell cycle without affecting DNA synthesis. The inhibition of cell division of B104 cells by ionomycin occurred within 24 h after stimulation. Because such a mode of action resembles that of anti-IgM antibodies, signals transduced by Ca2+ may be responsible for the inhibition of cell division of B104 cells by anti-IgM antibodies. Indeed, EGTA suppressed the inhibition of cell division of B104 cells caused not only by ionomycin, but also by anti-IgM antibody. Although TPA itself did not have any ability to promote the growth of B104 cells, it could cancel the inhibition of cell division of B104 cells by ionomycin and increase the proportion of B104 cells entering into the M phase of the cell cycle. Staphylococcus aureus Cowan I causes the greatest proliferation of normal human peripheral blood B cells during the period from 48 to 72 h after stimulation. When ionomycin was added to S. aureus Cowan I-stimulated peripheral blood B cells at 48 h of culture, it inhibited cell division during this period without affecting DNA synthesis. In the presence of TPA, this activity of ionomycin was suppressed, and the proportion of M-phase cells increased. These results suggest that cell division of mature B cells is regulated by the signals mediated by Ca2+ and protein kinase C in a mode quite different from that of regulation of DNA synthesis.     

Increased formation and decreased resorption of bone in mice with elevated vitamin D receptor in mature cells of the osteoblastic lineage.

The microarchitecture of bone is regulated by complex interactions between the bone-forming and resorbing cells, and several compounds regulate both actions. For example, vitamin D, which is required for bone mineralization, also stimulates bone resorption. Transgenic mice overexpressing the vitamin D receptor solely in mature cells of the osteoblastic bone-forming lineage were generated to test the potential therapeutic value of shifting the balance of vitamin D activity in favor of bone formation. Cortical bone was 5% wider and 15% stronger in these mice due to a doubling of periosteal mineral apposition rate without altered body weight or calcium homeostatic hormone levels. A 20% increase in trabecular bone volume in transgenic vertebrae was also observed, unexpectedly associated with a 30% reduction in resorption surface rather than greater bone formation. These findings indicate anabolic vitamin D activity in bone and identify a previously unknown pathway from mature osteoblastic cells to inhibit osteoclastic bone resorption, counterbalancing the known stimulatory action through immature osteoblastic cells. A therapeutic approach that both stimulates cortical anabolic and inhibits trabecular resorptive pathways would be ideal for treatment of osteoporosis and other osteopenic disorders.     

Immunoglobulin-containing cells in chick embryo urogenital tissues: a new site for early B lineage cells in endothermic vertebrates.

We have employed histological and immunofluorescent staining procedures in order to characterize the distribution of mu + B lineage cells in tissue sections prepared from developing chicken embryo urogenital tissues (UGTs) between 14 and 21 days of incubation. B lineage cells were observed in tissue sections prepared from developing UGTs, especially the mesonephros and its associated tissue, throughout the sample period. The highest densities of mu + B lineage cells were observed in tissue sections prepared from 18 day embryos. The mu + UGT cells were distributed singly and in clusters in subcapsular regions and within the peritubular interstitium of the mesonephros. These observations (1) are consistent with those which suggest nonbursal site(s) for origin of cells in B lineage, (2) may help account for the varying effects of embryonic caudectomy performed between the second and third days of incubation and surgical bursectomy performed close to hatching, (3) may help provide new insights on the effects of sex hormones on B cell development, and (4) suggest fundamental ontogenetic and phylogenetic similarities between developing vertebrate immune systems.     

Identification of a rabbit B cell alloantigen with an antiserum produced by alloimmunization with thymus cells.

Alloimmunization with rabbit thymus cells resulted in an antiserum (anti-Rly) which was shown to react with rabbit lymphocytes by an indirect rosette technique. The titration curve obtained with dilutions of anti-Rly antiserum on lymph node cells revealed two plateaus indicating that the antiserum was multispecific; at low dilutions of antiserum, within the first plateau, both B and T cells were rosetted whereas at high dilutions, within the second plateau, only B cells were rosetted. The antigen detected at high dilution was designated LB-1 (lymphocyte B cell alloantigen 1). The evidence that the cells identified within the second plateau are B cells is as follows: 1) simultaneous enumeration of LB-1⁺ and Ig⁺ (B) cells by use of distinguishable erythrocytes (sheep and human) as indicator cells revealed that of the 53% rosettes observed, essentially all (51%) were mixed rosettes containing both erythrocytes whereas simultaneous enumeration of LB-1⁺ and T⁺ cells (identified by anti-T cell antiserum) showed essentially no mixed rosettes (less than 2%); 2) approximately 80% of purified Ig⁺ (B) cells were identified as LB-1⁺ cells whereas essentially no (< 1%) purified T cells could be detected as LB-1⁺; 3) the percentages of LB-1⁺ cells and Ig⁺ cells were both reciprocal to the precentages of T⁺ cells identified in various lymphoid organs except for bone marrow; 4) the removal of LB-1⁺ cells from spleen cells of rabbits immunized with sheep red blood cells resulted in a depletion (42–71%) of direct plaque forming cells (PFC). Since the percentages of bone marrow cells rosetted using anti-LB-1 antiserum (approximately 70%) was much greater than the percentage rosetted using anti-Ig (approximately 10%), it appears that the anti-LB-1 antiserum is not directed against an Ig allotype. The titration curves of the anti-Rly antiserum on peripheral blood lymphocytes of a large rabbit family suggested that the LB-1 antigen on B cells is an alloantigen probably inherited in simple Mendelian fashion. Adsorption studies indicated that the LB-1 antigen on B cells is not detectable on brain, liver, kidney or erythrocytes.