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International Immunology, Vol. 13, No. 12, 1501-1514, December 2001
© 2001 Japanese Society for Immunology

xid mice reveal the interplay of homeostasis and Bruton's tyrosine kinase-mediated selection at multiple stages of B cell development

Michael P. Cancro, Alex P. Sah, Sherri L. Levy, David M. Allman, Madelyn R. Schmidt1 and Robert T. Woodland1

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 36th and Hamilton Walk, Philadelphia, PA 19104-6082, USA
1 Department of Molecular Genetics and Microbiology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01655, USA

Correspondence to: M. P. Cancro


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Human X-linked agammaglobulinemia (XLA) and murine X-linked immune defect (XID) are both immunodeficiencies mediated by mutations in Bruton's tyrosine kinase (Btk), yet the developmental stage(s) affected remain controversial. To further refine the placement of the XID defect(s), we used bromodeoxyuridine labeling to determine turnover, production and transition rates of developing B cell subsets in normal, xid and xid mice expressing a human Bcl-2 transgene (xid/bcl-2). We find the xid mutation manifest at two stages of B cell development. The first is early, reducing pre-B cell production by restricting pro-B to pre-B cell transit. Surprisingly, this impairment is offset by increased survival of cells progressing from the pre- to immature B cell pool, suggesting that Btk-independent homeostatic mechanisms act to maintain this compartment. The second point of action is late, substantially reducing mature B cell production. Together, these findings reconcile apparent discrepancies in the developmental stage affected by the murine versus human lesions and suggest previously unappreciated homeostatic processes that act at the pre-B to immature B cell transition. Finally, Btk likely functions differently at these two checkpoints, since ectopic Bcl-2 expression fails to directly complement the early xid lesion, yet reverses the defect impeding final B cell maturation.

Keywords: apoptosis, differentiation, homeostasis, immune deficiency, lymphocyte


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
BCR expression is a sequential process necessary for B cell maturation and survival (1–3). Signal transduction proteins Ig{alpha} and Igß are first expressed on the surface of pro-B cells associated with calnexin (4). In pre-B cells, newly synthesized H chains combine with the Vpre-B/{lambda}5 surrogate L chain to produce a functional surface receptor, the pre-BCR (5–9). Finally, in immature B cells a productively rearranged L chain joins with H chain and the Ig{alpha}/Igß heterodimer to produce a clonally restricted BCR. At each developmental stage, successful receptor expression is monitored by receptor-mediated signaling processes (10,11). Signals delivered via the Ig{alpha}/Igß heterodimer on pro-B cells induce competence for V to DJ rearrangement, whereas signaling through the pre-BCR promotes proliferation, allelic exclusion, L chain rearrangement and developmental progression through the pre-B cell stage (12). BCR signaling in immature cells mediates positive or negative selection, exit from the marrow, and subsequent entry into the long-lived mature B cell pool (13,14). Finally, ligand-mediated BCR signaling promotes the survival of quiescent mature cells (15), and initiates B cell proliferation and differentiation.

Since receptor-mediated signals are critical throughout a B cell's life history, it is not surprising that disruption of genes encoding signal transduction molecules can impair B cell development, selection and function (reviewed in 16). For example, Bruton's tyrosine kinase (Btk), a cytoplasmic protein tyrosine kinase mapping to the X chromosome at Xq22, is a signaling molecule critical for human B cell development. Male patients with btk mutations have a severe B cell immunodeficiency—X-linked agammaglobulinemia (XLA), that is characterized by reduced numbers of mature circulating B cells, diminished serum Ig levels and disrupted secondary lymphoid architecture (reviewed in 17). In XLA patients, the commitment of hematopoietic stem cells to the B cell lineage seems unimpaired, but B cell development is arrested at the cytoplasmic µ+ pre-B cell stage. The severity of the block is variable, as some patients have near normal numbers of pre-B cells. Nevertheless, these pre-B cells are defective in their proliferative capacity, accounting for the universal paucity of mature B cells in the periphery of XLA patients (18).

Mice also have a B lineage-specific, X-linked immune defect (XID) originally described in the CBA/N strain. These xid mice have a missense mutation at a conserved arginine residue within the Btk pleckstrin homology domain (19,20). Although btk is affected in both diseases, B cell depletion in murine XID is less severe than that seen in human XLA. Mice bearing xid have a half to a third the conventional follicular B cells (B2 cells) of normal mice, and a severe reduction of the B1a and B1b subpopulations (CD5+, B220+; MAC1+, B220+ respectively) that predominate in the peritoneum (21,22). Peripheral B cells from xid mice are functionally impaired; they are unresponsive to activation by mitogenic anti-Ig and by TI-2 antigens, such as haptenated Ficoll and pneumococcal polysaccharide (23,24). Moreover, xid B cells show a high rate of spontaneous apoptosis ex vivo and a significantly lower amount of endogenous Bcl-2 than is found in B cells from normal donors (25,26). The dysregulation of pathways that normally protect against apoptosis might thus contribute to the reduced pool of mature peripheral B cells in xid mice. The finding that ectopic expression of anti-apoptotic genes bcl-2 or bcl-xL stabilizes and preferentially expands this subpopulation in xid mice is consistent with this view (25,27).

While the XLA phenotype places the btk mutation's site of action at the pro-B to pre-B transition, establishing a site of action for the murine btk mutation has proven more difficult and controversial. Btk is involved in multiple signaling pathways including those for cytokines IL-5, IL-10, the mitogenic ectoenzyme CD38, the Toll-like receptor RP105 and the BCR (11,28–31). Deficiencies in these signaling pathways are known to inhibit the efficient activation of peripheral xid B cells. Btk signaling defects may also affect the late stages of B cell development. For instance, xid mice exhibit a higher than normal proportion of immature B cells [sIgMhi, sIgDlo, heat stable antigen (HSA)hi] in the spleen and reduced numbers of mature recirculating lymph node B cells, supporting the contention that XID restricts entry of immature peripheral B cells into the long-lived mature B cell pool (26,32,33). In contrast, the existence of early developmental defects in the bone marrow (BM) has been minimized by a lack of striking numerical differences in BM subpopulations when xid or btk knockout mice are compared to wild-type (34–36). Nevertheless, because btk is expressed at all stages of B cell development (37), the possibility remains that consequences of Btk signaling defects are also manifest at an early stage of B cell development in the BM. Indeed, it has recently been reported that mice deficient in both Tec and Btk have a block at the pre-B cell stage of development (38).

To rigorously define the sites of Btk function, we have examined the size, renewal and production rates within B lineage subsets in xid and wild-type mice using bromodeoxyuridine (BrdU) labeling. We also examined xid mice bearing a human bcl-2 transgene (Tg) (xid/bcl-2) (25), reasoning that ectopic expression of Bcl-2 might compensate for defects in apoptosis regulation arising because of Btk signaling deficiencies. Furthermore, because xid/bcl-2 transgenics have greatly increased numbers of recirculating mature (IgMlo, IgDhi) follicular B cells (25), a subpopulation deficient in xid mice, we applied these analytical methods to determine the developmental and/or peripheral changes responsible for follicular B cell reconstitution.

Our results show that xid produces significant cell losses at the early pre-B cell stage, thus placing the earliest manifestation of murine XID at the same developmental stage as human XLA. Surprisingly, we find near wild-type numbers of immature xid B cells in the marrow and periphery, likely reflecting the action of compensatory homeostatic mechanisms controlling the magnitude of the immature B cell pool. Consistent with the findings of others (26,32,33), we also observe a btk-mediated deficit in the immature to mature peripheral B cell transition, which reduces the rate of mature B cell production and thus the number of mature B cells. Ectopic Bcl-2 expression significantly and selectively increases the numbers of marrow pre-B cells, as well as both immature and mature peripheral B cells in xid/bcl-2 mice. The restoration of pre-B cells to wild-type numbers reflects prolonged survival within this compartment rather than the reversal of the xid lesion, because the pro-B to pre-B transition remains impaired. In contrast, ectopic Bcl-2 directly counteracts the peripheral xid lesion by extending the lifespan of both immature and mature peripheral B cells, facilitating their entry into and maintenance within the long-lived B cell pool.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
CBA/Ca (normal) and CBA/N (xid) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) or from the small animal production unit of the National Cancer Institute (Frederick, MD). Normal mice expressing a human (hu) bcl-2 Tg were originally obtained from Dr Stanley Korsmeyer (Washington University Medical School, St Louis, MO) and were maintained at the University of Massachusetts Medical Center by passage on a CBA/Ca background. Mice co-expressing the CBA/N defect and hubcl-2 (xid/bcl-2) were produced by mating CBA/N females with bcl-2 Tg+ males from CBA/NxCBA/Ca/bcl-2 crosses. Tg+ mice were identified by a PCR assay using DNA from tail biopsies prepared with a Qiagen DNA purification kit. PCR reaction produced a 304-bp fragment, using primers specific for hubcl-2 (sense: 5'-GGATGTTCTGTGCCTGTAAAC-3' and antisense: 5'-GGTCCATCTGAAGTTCCCAACTC-3'). For these experiments, both male and female mice were used. All animal husbandry and procedures were carried out in accordance with the Animal Welfare Act.

Lymphocyte suspensions
Lymphocyte suspensions were prepared as described previously (39,40). Briefly, BM cells were obtained from the two hind limbs of donor animals, and prepared by flushing the femurs and tibias with cold medium and aspirating the cell suspension using sequentially smaller bore needles. Spleen cell suspensions were prepared by crushing spleens between frosted glass slides and filtering the suspension through nylon monofilament cloth to remove debris. Erythrocytes were depleted by incubation in either Gey's solution or ammonium chloride–Tris.

Antibodies to cell-surface antigen and immunofluorescent analyses
The following reagents were purchased from PharMingen (San Diego, CA): phycoerythrin (PE)- and FITC-labeled anti-CD24 (HSA) (M1/69); PE-labeled anti-CD43 (leukosialin) (S7); allophycocyanin- and PE-labeled anti-CD45R (B220) (RA3-6B2). Biotin-labeled goat anti-mouse IgM, PE-labeled anti-IgD (SBA-1) and streptavidin–alkaline phosphatase were purchased from Southern Biotechnology Associates (Birmingham, AL). FITC-labeled anti-BrdU (B44) was purchased from Becton Dickinson (San Jose, CA). Cell-surface staining was done as described (39,41). Species- and isotype-appropriate controls were employed in all cytofluorimetric analyses.

Continuous BrdU labeling in vivo
The rate of continuous in vivo BrdU labeling was assessed as previously described (39–41). Mice were injected i.p. with 0.6 mg BrdU (Sigma, St Louis, MO) in 0.2 ml PBS at 12-h intervals for the duration of each experiment. Cells from BrdU-treated mice were stained for appropriate surface markers (PE, allophycocyanin and biotin-conjugated antibodies followed by Red670–streptavidin) as described above and washed with cold PBS. BrdU incorporation was analyzed according to previously published procedures. Briefly, the cells were permeabilized by dropwise addition of ice-cold 95% ethanol, washed, and then fixed in PBS with 1% paraformaldehyde and 0.05% Tween 20. The cells were then incubated in buffer plus 100 U/ml DNase to partially degrade and denature their chromatin. Finally, the cells were stained with FITC-labeled mAb to BrdU. Cytometric analyses were performed by gating on all nucleated cells.

Analysis of labeling kinetics within defined B cell subsets
For each mouse, the percentage of BrdU-labeled cells in each subset was measured by cytofluorimetry and multiplied by the total cells in the subset to give the number of labeled cells. The mean ± SD values for these percentages and numbers were plotted as a function of time, and least-squares regression analyses done to obtain the turnover and production rates respectively.

Production and renewal rates were calculated as previously described. Renewal rate is defined here as the percentage of a pool replaced per unit time by newly formed (labeled) cells. Production rate is the absolute number of newly formed cells generated within a pool per unit time. Briefly, the percentage of cells labeled at various days after the onset of BrdU treatment was determined by FACS and the renewal rates were then calculated by determining the slope of the percentage labeled versus time (percent per day) by linear regression. The absolute numbers of cells labeled were determined by multiplying the total cells in the subset by the proportion labeled and the production rate calculated as the slope of the regression equation for the number of cell labeled versus time. In the analyses of marrow subsets, we have expressed the data as cells per two hind limbs rather than to normalize the data based on total marrow cell estimates, because the total marrow nucleated cells in each strain of mouse used herein differ. Regression analyses were limited to the linear portion of the labeling plots, as determined by inspection, correlation coefficient and analysis of residuals. Generally, the labeling of newly forming marrow subsets in normal mice is linear until 3 days following onset of BrdU treatment, after which the curves plateau. In some mouse strains studied here, unusually slow proportional labeling rates were observed such that linear labeling kinetics persists longer, affording inclusion of additional points. The labeling of marrow subsets has been assumed to originate at the zero time point. While forcing the marrow regression lines through zero influences the exact numeric result of the calculations, it does not affect the relationships between treatment groups. Further, this convention minimizes differences between groups, affording the most conservative assessment. In the analysis of splenic subsets, no assumption of origin was made.

Cell cycle analysis
Cell cycle progression in BM subpopulations from CBA/Ca, CBA/N, normal/bcl-2 and xid/bcl-2 donors was determined by propidium iodide staining and FACS analysis. Developmentally distinct subpopulations were identified by staining with allophycocyanin-labeled-B220 (RA3-6B2), FITC-labeled CD43 (S7), biotin-labeled HSA (30F1), PE-labeled IgM (331.31) and Texas Red–streptavidin. Cells (1x105) from each population were sorted directly into cold 95% ethanol using a FACStar Plus (Becton Dickinson), incubated overnight at –20°C, then centrifuged and stained with PBS containing 1% glucose, 50 µg/ml RNase H and 50 µg/ml propidium iodide for 30 min at room temperature before analysis on a Becton Dickinson FACScan.

Determination of huBcl-2 expression in BM populations
Pro-B and pre-B cell populations from BM cells of xid/bcl-2 donors were purified by electronic sorting. Bone marrow cells were incubated with biotinylated anti-µ and anti-{delta} (Southern Biotechnology Associates), FITC-labeled anti-B220 (Caltag), PE-labeled anti-CD43 (PharMingen) and TriColor-labeled streptavidin (Caltag). TriColor+ cells were gated out and pro-B (B220+, CD43+, sIgM-) and pre-B (B220+, CD43-, sIgM-) positively selected. Immature B cells (sIgM+, slgD-) and mature B cells (sIgM+, sIgD+) were positively selected from a separate BM population stained with FITC-labeled anti-µ and PE-labeled anti-{delta}. Purity of isolated fractions was determined by flow cytometry and was >95%.

FACS-sorted BM cell populations were washed, pelleted and resuspended in lysis buffer (0.5% Nonidet P-40, 0.5% deoxycholate and 50 mM Tris, pH 8.0) supplemented with protease inhibitors as previously described (25,42). Cell extracts were also prepared from Percoll-purified small resting splenic B cells taken from hubcl-2 Tg+ and Tg- xid donors. The relative amount of huBcl-2 transgene in BM cell fractions was quantified by Western blotting as previously described (25), using a monoclonal anti-huBcl-2 antibody (6C8), generously provided by Dr S. Korsmeyer, that recognizes human but not murine Bcl-2 (43,44). Briefly, a huBcl-2 standard curve was prepared by serially diluting xid/bcl-2 Tg+ spleen B cell extracts with extracts from non-transgenic xid B cells to give a final concentration of 106 cell equivalents per lane. Likewise, cell extracts from 3x105 FACS separated BM cell fractions were diluted with the non-transgenic spleen B cell extract to give a final concentration of 106 cell equivalents for each BM subpopulation. Samples and standards were separated using SDS–PAGE (12.5%) and electrophoretic transfer to Immobilon-P. Membranes were sequentially treated with biotinylated anti-hamster Ig and streptavidin-conjugated horseradish peroxidase and developed using chemiluminescence (ECL; Amersham, Boston, MA). huBcl-2 concentration was determined for BM subpopulations, and compared to huBcl-2 in peripheral B cells by densitometry of multiple exposures on X-Omat film and reference to a serially diluted xid/bcl-2 spleen B cell standard.

Spontaneous apoptosis in BM cell cultures
Survival kinetics of FACS isolated pro-, pre-, immature and mature BM B cells from xid and xid/bcl-2 donors were determined for cultured cells. Thirty thousand cells from each subpopulation were cultured in 96-well round-bottomed tissue culture dishes in 100 µl of complete medium containing RPMI 1640, 10% FCS, 2 mM glutamine, 50 µM 2-mercaptoethanol, 100 µg/ml streptomycin, 10 µg/ml penicillin and 1xMEM non-essential amino acids. Cell viability was determined daily for 5 days by Trypan dye exclusion.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Enumeration of nucleated cells in the spleen and marrow of normal, xid and xid/bcl-2 mice
The number of nucleated cells recovered from the marrow of two hind limbs was determined for CBA/Ca (normal), CBA/N (xid) and xid/bcl-2 Tg mice (Fig. 1AGo, inset). The size of the marrow compartment is similar in xids and normals, 42x106 versus 38x106 respectively. In contrast, the number of cells recovered from xid/bcl-2 mice was consistently and significantly higher (64x106) than both xids and normals (P < 0.01).



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  		Fig. 1. Magnitude of B lineage progenitor pools in CBA mice. BM (A) or spleens (B) were harvested as described in Methods from either CBA/Ca (normal) mice (solid bars), CBA/N (xid) mice (open bars) or CBA/Nbcl-2 Tg+ (xid/bcl-2) mice (hatched bars). Total numbers of nucleated cells recovered from hind limbs or spleens are shown as insets. Representative gatings on CBA/Ca cells are shown to the left in each panel. Marrow cells were stained for sIgM, CD43 and B220, and splenocytes stained for sIgM, HSA, B220 and IgD as described in Methods. (A) Newly forming B cells and mature recirculating cells in the BM were identified as B220lo and B220hi respectively. The proportions of pro-B, pre-B and immature B subsets were determined by further analyzing cells within the B220lo gate for IgM and CD43, then multiplied by the total cells obtained from two hind limbs to yield cell numbers. (B) The proportions of immature and mature peripheral B cells were determined by analyzing B220+ cells for IgM, IgD and HSA (not shown), and their numbers calculated similarly. Bars show means ± SD for at least 15 mice of each strain.

 
Total nucleated spleen cells also differed among the three groups (Fig. 1BGo, inset). On average, 100x106 splenocytes were recovered from normal mice, significantly more (P < 0.01) than the average of 62x106 obtained from xid mice. As found previously (25), the ectopic expression of Bcl-2 substantially increases spleen cellularity in xid/bcl-2 mice (206 ± 49x106) relative to both normal and xid mice (P < 0.01).

The marrow and splenic B cell subsets in normal, xid and xid/bcl-2 mice
The proportional representation of each B lineage subset in the marrow and spleen was determined by flow cytometry, and the numbers of cells in each subset calculated based on the total cell recoveries (Fig. 1AGo). Surface expression of sIgD, sIgM, B220 and CD43 was used to discriminate marrow B cell differentiation subsets as previously described (45). Representative gatings on normal (CBA/Ca) marrow are shown at left.

The average number of pro-B cells (B220lo, CD43+, sIgM-) is similar in all three strains (Fig. 1AGo): 1.2, 1.3 and 1.5x106 cells for normal, xid and xid/bcl-2 respectively. This suggests that neither xid nor introduction of the bcl-2 transgene alter the efficiency of B lineage commitment. In contrast, differences in the pre-B cell subset (B220+, CD43-, sIgM-) were readily apparent. Marrow from xid mice yielded a mean of 3.2x106 pre-B cells, significantly fewer (P < 0.01) than the 6.5x106 found in normal mice. Introducing bcl-2 Tg increased pre-B cell numbers in xid/bcl-2 donors to a mean of 6.3x106 cells, similar to wild-type. Finally, the 1.1x106 immature marrow B cells (B220+, IgM+, IgD-) in xid mice was similar to the 1.5 and 1.3x106 observed in normal and xid/bcl-2 mice respectively, inasmuch as the small but consistent differences seen were not statistically significant. Thus, despite the reduction in pre-B cells induced by xid, compensatory homeostatic mechanisms appear to have restored the immature B cell compartment in xid mice. Recirculating mature B cells (B220hi, IgM+/–, IgD+) in the xid marrow were only half the number found in normals (0.6 ± 0.46 versus 1.2 ± 0.45x106 respectively), whereas xid/bcl-2 Tg mice had 14 times more (17x106). This subset largely accounts for the higher cellularity of xid/bcl-2 marrow.

Newly emerging B cells exit the BM and spend several days in the periphery, where they remain functionally and phenotypically immature before final maturation and entry to the long-lived mature B cell pool (32,33,39,41). It has been reported that xid mice have a higher ratio of immature to mature B cells than normals and this imbalance has been proposed to account for some of the functional abnormalities of peripheral xid B cells (26). Immature and mature peripheral B cells can be distinguished by the differential expression of HSA (CD24), B220 (CD45), sIgM and sIgD, allowing splenic B cell subsets to be enumerated in each of the three strains by surface antigen phenotype (Fig. 1BGo). Immature splenic xid B cells (B220+, HSAhi, IgM+, IgD-) were reduced in absolute number relative to wild-type, although this difference did not reach statistical significance. In contrast, the number of immature splenic B cells in xid/bcl-2 mice was consistently and significantly higher than that found in either xid or wild-type mice.

The numbers of mature splenic B cells differed significantly among all strains. As shown previously by us and others, xid mice had 3- to 5-fold fewer mature splenic B cells than normal mice, 16 ± 12.4 versus 46 ± 8.8x106 (Fig. 1BGo). Moreover, xid/bcl-2 mice generally had more than twice the number of mature B cells found in normal mice and 10 times the number found in xids.

Transgene expression in BM subsets
One explanation for our finding that pre-B cells are the only resident subpopulation increased in xid/bcl-2 marrow is that ectopic expression of Bcl-2 has a selective protective effect. Although it was shown previously that the Ig–huBcl-2 construct used for our transgenics is efficiently expressed in all the marrow B cell subpopulations of transgenic normal mice (46), the pattern of expression of huBcl-2 in xid/bcl-2 transgenics has not been established. To determine if biased Tg expression was the basis for selective protection, we quantitated Tg protein in FACS-sorted pro-, pre-, immature and recirculating mature marrow B cells from xid/bcl-2 mice. Whole-cell extracts from fixed numbers of purified BM subpopulations were analyzed and the relative amount of huBcl-2 determined (see Methods) using a monoclonal anti-huBcl-2 antibody (6C8) that recognizes human but not murine Bcl-2 (43). The results, in Fig. 2Go, show huBcl-2 protein is produced by each BM subpopulation, and, with the exception of pro-B cells, the amount of Tg product was similar for marrow and splenic B cells. We also found ectopic Bcl-2 expression significantly retarded the rate of spontaneous apoptosis in culture, relative to xid controls, of all B cell subpopulations including pro-B cells (data not shown). This result is similar to what has been found for normal/bcl-2 transgenics (47). It is thus most likely that ectopic Bcl-2 increases cell numbers by reducing apoptotic cell loss, a conclusion supported directly in a subsequent section.



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  		Fig. 2. Quantitation of huBcl-2 protein in BM cells from xid/bcl-2 donors. (A) Titration of huBcl-2 protein in spleen cell extracts. Whole-cell extracts from serially diluted xid/bcl-2 spleen cells were mixed with non-Tg xid B cell extracts to give a final loading of 106 cell equivalents per lane. Extracts were separated by 12.5% PAGE, electrophoretically transferred and probed with mAb (6C8) specific for huBcl-2. A titration curve was generated from densitometry scans of the Western blots. (B) huBcl-2 protein in BM subpopulations. Bone marrow populations from xid/bcl-2 donors were FACS sorted, using the criteria described in Methods. Whole-cell extracts were prepared from each population and mixed with extracts of non-Tg xid spleen B cells to give a constant number of BM cells and a final loading of 106 cell equivalents per lane. Samples were processed as described in (A) and the amount of huBcl-2 relative to peripheral xid/bcl-2 B cells, determined for each subpopulation by reference to the spleen cell titration curve. (C) Specificity controls. There was no signal for huBcl-2 detected with 6C8 when cell extracts from106 non-Tg xid spleen B cells were analyzed. The signal generated from the non-specific (NS) binding of 6C8 and anti-hamster Ig to an unidentified protein in the spleen cell extracts is shown as a control for sample loading.

 
Turnover and production rates in BM B lineage subsets of normal versus xid mice
The reduction of pre-B cells by xid could result from reduced survival rates either within or immediately prior to this differentiation stage. Further, the relatively normal size of the immature marrow pool suggests that btk-independent compensatory mechanisms may intervene subsequent to the xid lesion. These may increase the proportion of pre-B cells transiting to the immature B cell stage or the longevity of cells within the immature B compartment. To distinguish among these possibilities, kinetic studies of the dynamics within each marrow and splenic B lineage subset were undertaken. Labeling studies with BrdU were performed over a 3-day time course, because >95% of all B220+ BM cells are labeled in normal young mice during this period. The BrdU treatment was not cytotoxic, since it changed neither the number of BM B cells recovered nor the proportions of newly formed versus mature B cells in a given strain (data not shown). Furthermore, within each strain, there was no strong or consistent difference in the total number of nucleated BM cells recovered during the course of the experiment.

Figure 3Go depicts the fraction of the total (upper panels) and absolute numbers (lower panels) for each B lineage subset labeled over time in normal and xid mice. By using the calculations described in Methods, the initial linear portions of these two sets of curves provide estimates of subset renewal and production rates respectively. Because of the differences in marrow cell numbers across the three strains studied, the absolute labeling rates are expressed in terms of the actual cells recovered from two hind limbs.



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  		Fig. 3. Kinetics of BrdU labeling within each B lineage progenitor pool in normal and xid mice. Normal mice (filled diamonds, dashed line) or xid mice (open squares, solid line) were injected with 0.6 mg of BrdU at 12-h intervals. BM was harvested at various times after labeling and stained for sIgM, CD43 and B220. The stained cells were permeabilized, fixed and then stained with FITC-labeled antibodies to BrdU as described in Methods. The proportion of BrdU-labeled cells (upper panels) was determined cytofluorimetrically and the absolute number of BrdU-labeled cells (lower panels) calculated by multiplying the proportion of labeled cells by the total cells obtained per two hind limbs. Labeling among pro-B, pre-B and immature B-cell subsets is shown in the left, middle and right plots respectively. Each point represents an individual mouse. Renewal and production rates were determined by least-squares regression on the linear portions of each data set and the predicted line from the regression equation is overlaid. These calculations are described in detail in Methods.

 
Both renewal and production rates of pro-B cells (B220lo, sIgM-, CD43+) were similar in normal and xid marrow (Fig. 3Go, left panels). In both strains, the pro-B compartment has a renewal rate of ~35%/day, corresponding to a production rate in the range of ~0.3x106 cells/day per two hind limbs (Table 1Go). This finding is in accord with the similar numbers of pro-B cells observed in normal and xid marrow, and further strengthens the conclusion that xid has little influence on B lineage commitment.


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  		Table 1. Magnitude and kinetics of bone marrow B cell subsets
 
In contrast, pre-B cell labeling kinetics of normal versus xid mice differ significantly (Fig. 3Go, middle panels). Normal adults generate ~ 2x106 pre-B cells/day, whereas xids produce only ~0.5x106 pre-B cells/day (Table 1Go). Nonetheless, the pre-B cell renewal rates are identical (35–36%/day), indicating that residency time within the pool is unchanged by xid (Table 1Go). This substantial reduction in production rate, coupled with relatively unchanged renewal rate, is consistent with the reduced numbers of pre-B cells seen in xid mice (Fig. 1AGo). Importantly, attrition within the pre-B cell pool is unaffected by xid, since increased attrition would yield a more rapid renewal rate. The reduction in pre-B cell formation coupled with the lack of increased attrition within the compartment suggests xid targets the transit of pro-B cells to pre-B cells.

Despite the 2-fold reduction in pre-B cell number, neither the renewal rate (26 versus 27% daily) nor production rate (0.28 versus 0.22x106 cells/day) of immature marrow B cells differs significantly between normal and xid mice (Fig. 3Go, right panels). This surprising finding indicates that a larger proportion of xid pre-B cells, relative to wild-type, succeed in differentiating to the immature compartment. Further, it accounts for the similar numbers of immature B cells observed in the BM and spleen (Fig. 1Go), and suggests that Btk-independent homeostatic mechanisms operate to maintain an immature compartment of relatively constant size.

Renewal and production rates in marrow B lineage subsets of bcl-2 transgenic xid mice
Ectopic expression of the bcl-2 Tg restores the pre-B cell compartment of xid/bcl-2 mice to the number found in wild-type mice (Fig. 1Go). This could reflect a reversal of the xid-mediated attrition at the pro-B to pre-B cell transition or instead indicate a change in the renewal rate of pre-B cells. These possibilities were distinguished by determining the renewal and production rates of each B lineage subset in xid/bcl-2 mice (Fig. 4Go).



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  		Fig. 4. Kinetics of BrdU labeling within each B lineage progenitor pool in xid and xid/bcl-2 Tg mice. xid mice (open squares, solid line) or xid/bcl-2 Tg mice (filled circles, dotted line) were processed as in Fig. 3Go. The proportion of BrdU-labeled cells (upper panels) was determined cytofluorimetrically and the absolute number of BrdU-labeled cells (lower panels) calculated as above. Labeling among pro-B, pre-B and immature B cell subsets is shown in the left, middle and right plots respectively. Each point represents an individual mouse. Production rates predicted by least-squares regression are overlaid for each set of points. Determinations of production and renewal rates were as described in Fig. 3Go and Methods.

 
The renewal and production rates of pro-B cells in xid/bcl-2 mice are similar to those determined for xid and normal mice (Fig. 4Go, left panels, and Table 1Go). This is not surprising since endogenous Bcl-2 is normally expressed within pro-B cells (48). In contrast, the absolute labeling rate of xid/bcl-2 pre-B cells was similar to that in xids but only 25–40% that seen in normals (Fig. 3Go, middle panels). This shows that ectopic Bcl-2 expression does not ameliorate xid-mediated cell loss at the pro-B to pre-B transition, because pre-B cell production rates equivalent to normal would have been seen if this were the case. Ectopic Bcl-2 expression does, however, change the dynamics within the compartment: pre-B cells in xid/bcl-2 transgenic mice were renewed at a rate of only 9%/day, which differs markedly from the 35%/day observed in both wild-type and xid mice (Table 1Go). At this rate it required ~6 days for transgenics to achieve 50% labeling (Fig. 4Go, middle panels) compared with ~36 h for either xid or normal, thus necessitating the extended labeling period used in the analysis. These results suggest that Bcl-2 restores the pre-B cell compartment by lengthening the survival and/or residency time within the pre-B cell pool, not by directly reversing the effects of xid per se.

The number of immature BM B cells in xid/bcl-2 transgenics was not increased relative to normals or xids (Fig. 1Go and Table 1Go), this despite the finding the renewal rate of these cells is reduced by 3-fold (to 10%/day) relative to wild-type (Table 1Go). This reduced renewal rate is counterbalanced by a 2-fold decrease in the daily production rate, thereby maintaining immature xid/bcl-2 BM B cells at wild-type numbers. These data, in toto, show that ectopic Bcl-2 expression lengthens survival of all marrow differentiation subsets subsequent to the pro-B cell stage.

Cell cycle analyses of marrow subpopulations
Two sets of experimental findings suggest multiple interpretations that could be resolved through cell cycle analysis. First, we found a reduced number and production rate of pre-B cells in xid mice. This could indicate either a btk-mediated impairment of the pro-B to pre-B cell transition or a normal transit but impaired replication of cells within the pre-B cell compartment. The latter possibility could be directly addressed by cell cycle analysis. Second, we found a reduced renewal rate for pre-B and immature marrow B cells in xid/bcl-2 transgenics. This could result either from inhibition of cellular attrition by Bcl-2 or from a slowing in the division rate, a phenomenon seen for several cell types expressing ectopic Bcl-2 (49,50). To evaluate these alternatives, we determined the fraction of cycling cells by propidium iodide staining of FACS purified pro-B cell (fractions B/C), large pre-B cells (fraction C'), small pre-B cells (fraction D) and immature B cells (fraction E) separated using the criteria established by Hardy (51). Pooled BM cells from groups of three normal, xid, normal/bcl-2 and xid/bcl-2 donors were used for the analysis.

A comparison of xid to wild-type BM cells (Fig. 5Go), showed no significant difference in the fraction of cycling cells in the pro-B (11 versus10%), large pre-B (54 versus 59%) or immature B (1.3 versus1.3%) cell subpopulations. The proportion of replicating cells in the small pre-B compartment did differ significantly between xid and wild-type mice (4 versus 11%, P < 0.005), but cell cycle differences within this relatively small fraction of replicating cells could not account for the 2-fold reduction in the absolute number of cells constituting the total xid pre-B cell compartment. As to the second point, the data in Fig. 5Go comparing xid/bcl-2 transgenics to xids show that ectopic expression of Bcl-2 does not reduce the fraction of cycling cells within any marrow B cell subpopulation. Indeed, with the exception of pro-B cells, all other developmental stages had a significant increase in the fraction of cycling cells. Consistent with a previous report (50), cycling cells were reduced in normal/bcl-2 donors relative to non-transgenic normal littermates, and this reduction was most significant for pro-B and small pre-B cells (P < 0.02 in each case). Overall, these data suggest that: (i) the reduction in the number of xid pre-B cells results from an impairment of pro-B cell transit to the pre-B cell stage rather than from significant reduction in replicative vigor within the early pre-B compartment, and (ii) the increased renewal rate of pre-B and immature marrow B cells in xid/bcl-2 transgenics most likely results from a Bcl-2-dependent reduction in B cell attrition within these compartments, rather than from a retardation in cell cycle progression.



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  		Fig. 5. Cell cycle analysis of BM subpopulations from xid and normal bcl-2 Tg+ and non-Tg littermates. Cell cycle progression in BM cells was analyzed by FACS using propidium iodide staining, of BM subpopulations distinguished by surface staining with antibodies to B220, sIgM, slgD, CD43 and HSA as described in Methods. Each set of data is the mean of three mice. All data include a SD, but some are too small to be distinguished.

 
Renewal and production rates of splenic B cell subsets in normal, xid and xid/bcl-2 mice
Several laboratories have reported an xid-mediated defect in peripheral B cell maturation (26,32–35). The marked expansion, in xid/bcl-2 transgenics (25), of a mature B cell subpopulation normally deficient in xid mice suggests this late btk-dependent maturational defect can be corrected by ectopic Bcl-2 expression. The mechanism by which this correction occurs has not been established. Bcl-2 may act simply to decrease the attrition of mature xid B cells or may provoke more subtle effects. To gain more insight regarding the action of ectopic Bcl-2 we extended our kinetic studies to B cells in the periphery, determining the magnitude, renewal rates and production rates of splenic immature (B220lo, HSAhi, IgM+) and mature (B220hi, HSAlo, IgM+, IgD+) B cell populations in normal, xid and xid/bcl-2 mice. These results are summarized in Table 2Go.


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  		Table 2. Magnitude and kinetics of splenic B cell subsets
 
The number, renewal rate and production rate of immature peripheral B cells did not differ significantly between xids and normals. Because the vast majority of labeled peripheral B cells are immigrants, rather than cells generated by division in the periphery, these data suggest that immature xid marrow B cells emigrate to the periphery efficiently, i.e. in numbers and with kinetics comparable to wild-type, and that Btk normally plays little role in this transit. In marked contrast, the production rate of mature splenic xid B cells is reduced 4-fold relative to wild-type, accounting, in large part, for their reduced steady-state number (Fig. 1Go and Table 2Go). Comparing the production rates of immature and mature B cells shows that <15% of immature xid peripheral B cells join the mature B cell pool, whereas ~50% make this transition in normal mice. Importantly, the renewal rate of the mature B cell pool is similar for both strains, suggesting that while xid perturbs recruitment into the long-lived mature peripheral pool, those xid B cells that successfully achieve this transit have a lifespan comparable to wild-type.

Both peripheral B cell compartments were significantly expanded in xid/bcl-2 mice relative to both xid and normal mice (Table 2Go). Immature splenic B cells were 4 and 7 times the number found in wild-type and xids respectively. Likewise, mature B cells were increased by >2-fold versus wild-type and 12-fold versus xid mice. For both populations, cellular increases were effected both by a reduction of the renewal rate and an increase in the production rate. Most noteworthy, however, is the finding that 50% of immature xid/bcl-2 B cells differentiate into mature B cells, an efficiency indistinguishable from normal mice. Thus a diminution of cellular attrition within the immature B cell subpopulation allowed a more efficient differentiation of these cells to the mature B cell compartment. These results strongly suggest the late-acting xid-mediated defect at the immature to mature peripheral B cell transition is directly ameliorated by ectopic Bcl-2 expression.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We have measured and compared the magnitude, production rates and renewal rates of B lineage subsets in the marrow and periphery of normal, xid and xid/bcl-2 mice. Our findings, summarized in Fig. 6Go, are noteworthy for several reasons. First, we have resolved a discrepancy in the placement of the btk defect as determined in mice and humans. We find the first manifestation of btk deficiency in xid mice is evident during the pro-B to pre-B cell transition, placing the initial effects of murine xid and human xla mutations at the same stage of B cell development. Although the bcl-2 Tg promotes lengthened survival in most B cell subsets, it does not abrogate the losses at the pro/pre-B interface mediated by xid. Second, immature B cells in the xid marrow are close to wild-type numbers despite reduced numbers of xid pre-B cells demonstrating previously unappreciated compensation mechanisms acting at this stage in development. Third, we show that immature B cells emigrate from the xid BM at wild-type rates and numbers, but once in the periphery their recruitment into the long-lived mature B cell pool is markedly impaired. This second consequence of the xid mutation is abrogated by ectopic Bcl-2 expression because both the rate of mature B cell production and the proportion of immature B cells recruited into this compartment reach wild-type levels.



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  		Fig. 6. Proposed model of xid-mediated lesions in murine B cell differentiation. The relative expression of surface markers used in this study is shown below the schematic of B cell differentiation. Proposed points of xid action are indicated and B cell subsets whose sizes are altered by xid are shaded.

 
Pre-B cell production is deficient in xid mice
The number of pro-B cells is similar in xid and wild-type mice, indicating commitment to the B cell lineage is unimpaired. Moreover, neither the number nor the renewal rate of xid pro-B cells was significantly affected by ectopic Bcl-2 expression. This finding is in accord with studies of normal bcl-2 and bcl-xL transgenics (52), and likely reflects the high amount of Bcl-2 normally expressed in pro-B cells (46). In contrast, xid pre-B cells were significantly reduced as a direct result of diminished pre-B cell production. We posit the basis for this reduction is that the btk defect restricts the number of pro-B cells that successfully transit to the pre-B cell stage of development. The majority (80%) of pre-B cells are quiescent and derived directly from a pool of large pre-B cells driven to proliferate by pre-BCR signaling (53). Accordingly, we considered the possibility that the replication of pre-B cells could be impaired by xid, as is found in human xla (18). Our cell cycle analyses showed, however, that the percentage of cycling pre-B cells was not significantly reduced in xid mice. Furthermore, the renewal rates within the pre-B compartment were the same as wild-type, showing xid B cells progressing to this developmental stage are robust and loss from within the compartment is not excessive. Therefore, the most likely explanation for the diminished number of xid pre-B cells is that fewer pro-B cells enter the pre-B cell pool. This demonstrates a role for cell signaling in the transition between pro-and pre-B cells and the participation of Btk at this developmental checkpoint.

Btk-dependent signals could be necessary for optimal pro-B cell differentiation and progression to the pre-B cell stage. It has been proposed that signals delivered to pro-B cells through Igß precede VH–DJH rearrangement and allow progression to the pre-B cell stage (4,12). It has not been determined if Btk-dependent pro-B cell signals are necessary for efficient Ig gene recombination, but it is noteworthy that VH repertoire utilization in xid mice is skewed, relative to wild-type, at the pre-B stage of development (54).

Ectopic Bcl-2 expression does not correct the impaired pro-B to pre-B cell transition
Introducing a hubcl-2 Tg onto the xid background restored the pre-B cell compartment to the wild-type level. The xid mutation was not directly ameliorated, however, because the primary defect, a reduced rate of xid pre-B cell production, was unchanged in xid/bcl-2 transgenics. Instead, ectopic Bcl-2 decreased the turnover rate of xid/bcl-2 pre-B cells >3-fold, compensating for reduced pre-B cell production and establishing a pre-B cell compartment of wild-type size. In normal mice the expression of either a bcl-xL or bcl-2 Tg also increases the size of the pre-B cell compartment (46,52,55) by rescue of cells normally lost to apoptosis (56,57). The alternative explanation, that the reduced rate of pre-B cell renewal reflected Bcl-2-mediated retardation of cell cycle progression (50), was excluded by our finding that the fraction of cycling cells in all xid/bcl-2 BM subpopulations was the same or greater than comparable populations in xid mice.

Support for an early placement of xid
Our data place the initial manifestation of the xid lesion at the pro-B to pre-B cell transition, the same site proposed for human xla (18,58,59). This early developmental perturbation is in addition to the more familiar late manifestation of xid (33–36,60) and is consistent with earlier suggestions of multiple sites of action for btk mutations in XLA (61). Our early placement of xid is supported by multiple lines of evidence. Tec and Btk are members of the Tec kinase family that are active during lymphocyte development, and have similar biochemical function (62). While Tec single knockouts have no discernable immune defects, Btk/Tec double-deficient mice have a severe B cell deficiency due to a partial block in B cell development at the pro-B to pre-B cell transition (38), strongly suggesting the requirement for Btk at this stage in development. Other genetic analyses are also consistent with this finding. The adapter protein BLNK, as well as the kinases Syk and phosphatidylinositol-3-kinase (PI3K), are directly involved in Btk activation and function (63), particularly the Btk-dependent sustained influx of calcium seen after BCR engagement (64,65). Targeted mutation of either BLNK or the p85{alpha} subunit of PI3K, yields mice whose phenotype resembles xid (66–68). Developmental abnormalities in these mice are first seen at the pro-B to pre-B transition, reducing the number of pre-B cells 2- to 4-fold. Lastly, the non-receptor protein tyrosine kinase Syk initially activates Btk after BCR engagement (63,69). Although disruption of syk is embryonic lethal, immune function can be assessed in Rag-2–/– mice reconstituted with syk–/– fetal liver. These chimeras show defects in B cell development first apparent after the pro-B cell stage, which again severely limits the production of pre-B cells (70,71).

Another approach used to establish early site(s) for Btk action was to inactivate the btk gene, and determine when and if Btk–/– B cells suffered a selective disadvantage as they developed in the same microenvironment with competing normal B cells. Kerner and colleagues (72) produced chimeric mice by introducing btk–/Y embryonic stem cells into B6 blastocysts. In these chimeric mice there was a striking reduction of btk–/– B cells at the pre-BII (B220+, IgM-, CD43-) stage of development, suggesting a defect at the pro-B to pre-B cell transition. Hendriks and colleagues inactivated btk by inserting a lacZ reporter in frame into the btk locus (73). They found that Btk-/LacZ+ B cells in btk–/+ female heterozygotes were first noticeably lost at the immature B cell stage, suggesting an impairment of the pre-B to immature B cell transition. The difference in the Hendriks finding and the other genetic studies cited may be due to the influence of non X-linked genes from the129 background. It has recently been shown that 129 sublines have xid-like defects that impair Btk-dependent B cell signaling and these signaling defects are exacerbated when the 129 gene(s) are co-expressed with a defective btk gene (74). In the Hendricks study 129-derived embryonic stem cells were used to produce the btk targeted LacZ knockin used and the mice derived therefrom, although crossed with B6, still contained a significant fraction of129 genes. It is thus possible that the Hendriks study compared two immunodeficient cell populations, rather than the xid to wild-type comparison attempted.

Our review of the available data supports our conclusion that the initial manifestation of the xid mutation is during B cell development at the pro-B to pre-B cell transition.

Immature B cells in the xid BM rebound
Although the xid lesion reduces the number of pre-B cells available, the immature BM population is maintained at near wild-type levels. This reflects enhanced proportional survival of pre-B cells that differentiate to immature B cells: 40% of xid B cells make this transition as opposed to <15% of wild-type. This suggests the operation of a homeostatic mechanism(s) that ensures an immature marrow compartment of relatively constant size. This view is further supported by our finding that while the renewal rate of pre-B and immature B cells is significantly slowed in xid/bcl-2 transgenics, the absolute number of immature marrow B cells is maintained at a wild-type level, because the fraction of pre-B cells that progress to immature B cells is reduced.

Regulation of immature xid B cells in the periphery
Btk apparently has little effect on the egress of immature B cells to the periphery, as the production rate and number of immature splenic B cells is not significantly different in normal and xid mice. This finding is in agreement with previous reports showing BrdU-labeled B cells in CBA/Ca and CBA/N mice arrive in the spleen in approximately equal numbers (32), producing immature splenic B cell populations of similar size (32,33). It has been suggested that xid B cells emigrate from the BM at significantly higher numbers than wild-type (75), a finding that we fail to see. Indeed, one would predict that the immature B cell compartment in xids should be significantly larger if marrow egress were markedly increased, unless the turnover rate within this compartment was accelerated. It is noteworthy that neither Cariappa and colleagues (75) nor we have noted an increase in immature xid B cell numbers or turnover.

In contrast to marrow cells, immature B cells in the xid/bcl-2 spleen are significantly increased. This comes about because of an increased cell production rate combined with a decreased cell turnover rate. A similar expansion of immature splenic B cells is seen in normal bcl-2 Tg mice (76). These results suggest that the homeostatic regulation of the marrow and splenic compartments differs. Immature B cells in transit to or in the spleen are thought to be most susceptible to apoptosis because they have left the protective microenvironment of the BM (77,78) and/or perhaps because of increased BCR expression. Our findings with xid/bcl-2 mice are consistent with the notion that immature xid B cells are removed aggressively in the periphery by apoptosis because ectopic expression of Bcl-2 can attenuate this loss. This suggests that the intensity of BCR signaling in xid mice, although reduced, is still sufficient to delete autoreactive cells or, alternatively, that cell loss by neglect is also a consequence of deficient BCR signaling. The former possibility seems more likely as it can readily be envisioned how suppression of apoptosis by Bcl-2 could promote additional opportunities to divert receptor specificity away from autoreactivity but not how receptors susceptible to positive selection would be generated. It is also noteworthy that there are no examples of xid mice preferentially making autoreactive antibodies, indeed the xid mutation suppresses such autoantibody production (79–81).

Bcl-2 rescues xid B cells normally lost at the immature to mature B cell transition
The major difference we noted in peripheral B lymphocyte populations of normal and xid mice was the severe restriction on xid B cells progressing to the mature stage of B cell development. While 50% of normal immature splenic B cells make this transition, <15% of xid B cells did so. The renewal rates we determined suggest, however, that xid B cells making this transition are relatively robust. This late-stage developmental block has been described repeatedly (33–35, 60) and could reflect either Btk-facilitated positive selection into the long-lived B cell pool or potentiation of signals requisite for the final stages of B cell maturation. Ectopic Bcl-2 expression directly corrects this late stage manifestation of xid, as comparison of production rates reveals that the proportion of immature B cells joining the long-lived pool increases to the level found in wild-type mice. These mature xid B cells have properties of normal mature B cells, including active recirculation and high levels of sIgD. If one assumes the signals promoting selection into the long-lived pool are cumulative or that intracellular second messengers can be acquired over time, then Bcl-2 may extend the survival of immature B cells sufficiently to effect this transition.


   Acknowledgments
 
The authors acknowledge the valuable expertise and guidance of Mr Hank Pletcher and Ms Danielle DeHoratius at the University of Pennsylvania Cancer Center Flow Cytometry Facility. We also wish to thank Drs Rachel Gerstein and Janet Stavnezer, University of Massachuesetts Medical School, and Dr Joan Press, Brandeis University for their critical review of the manuscript and thoughtful comments. This work was supported in part by a grant from the Lucille P. Markey Charitable trust to M. P. C.; and USPHS grants AG15623 to M. P. C., and AI30890, AI41054, AG15178 and an Institutional Diabetes and Endocrinology Research Center Grant from the National Institutes of Health (DK32520) to R. T. W.


   Abbreviations
 
BM bone marrow
BrdU bromodeoxyuridine
Btk Bruton's tyrosine kinase
HSA heat stable antigen
Hu human
PE phycoerythrin
PI3K phosphatidylinositol-3-kinase
Tg transgene
XID X-linked immune defect
XLA X-linked agammaglobulinemia

   Notes
 
Transmitting editor: P. Kincade

Received 6 August 2001, accepted 29 August 2001.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
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