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Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It was established by Hardy et al. (7, 8) that peripheral B cells can be categorized into fractions I, II, and III based on the relative levels of IgM and IgD, and that fraction I (IgDhighIgMlow) cells are considerably diminished in the Xid mouse. A similar phenotype has been described in Btk3 null (9, 10, 11), and CD45 null mice (12). Btk is known to be downstream of the B cell receptor (13, 14, 15), and CD45 is essential for Ag receptor signaling (16). In contrast, CD22 plays an inhibitory role in B cell receptor signaling; in CD22 null mice fraction I cells are more abundant than in wild-type mice (17, 18, 19). These findings have lent support to the view that positive selection or ligand-independent signaling via the Ag receptor might drive the formation or survival of fraction I cells. The phenotypes of the Xid, Btk null, CD45 null, and CD22 null mice all indirectly suggest that the development or persistence of fraction I cells might be regulated by Ag receptor signaling.
The emigration of recently generated B cells from the bone marrow to the periphery has been demonstrated to depend on signals from the B cell receptor (5). Only a proportion of newly generated B cells actually reach the periphery (3, 20, 21). How much of this cell loss is attributable to positive or negative selection, and whether cells are lost in the bone marrow or soon after entering peripheral organs remain unclear. Although earlier evidence from transgenic models suggests that self-reactive immature B cells may be eliminated during development (22, 23), it is also possible that few developing cells are actually deleted by self Ags; receptor editing may represent a major mechanism for containing self reactivity during early development (24, 25, 26, 27). Once B cells leave the bone marrow they migrate through the T cell areas of peripheral lymphoid organs on their way to follicular niches. It is possible that the Syk tyrosine kinase is required for emigration from the bone marrow and for the process of follicular entry (28).
Studies on B cell life span have suggested the existence of a short-lived population of B cells with a half-life of 3–4 days and a longer lived population with a half-life of about 6 wk (29, 30, 31). The recent studies of Lam et al. (6) have suggested that once B cells enter the periphery, signals from the Ag receptor are required for the maintenance of long-lived B cells. The life span in vivo of B cells in Xid mice is reduced when they are placed in competition with normal B cells (32, 33), and these results are supported by studies indicating a decreased viability of Xid B cells in vitro (34). It is possible that Btk participates in the Ag receptor-mediated peripheral persistence of naive B lymphocytes.
We ask whether the relative absence of only fraction I cells in mice lacking Btk represented a defect in the maturation from fraction II to fraction I or a defect in the survival specifically of fraction I cells. Our results suggest that a Btk-dependent maintenance checkpoint during naive B cell maturation is manifest specifically in fraction I B cells. As a presumed consequence of this maintenance defect, the emigration of newly generated B cells into the periphery occurs in an accelerated fashion in Xid mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to twelve-week-old CBA/Ca and CBA/N mice from The Jackson Laboratory (Bar Harbor, ME) were used in this study. Age- and sex-matched mice were used in all experiments.
Bromodeoxyuridine (BrdU) labeling
Pulse labeling with BrdU was performed by injecting mice i.p. with a single 5-mg dose of BrdU (Sigma) dissolved in 0.5 ml of sterile PBS. All injections were timed to permit the simultaneous harvest of lymphocytes from multiple groups of mice at a fixed time (0800 h). Continuous labeling was performed by feeding mice 0.25 mg/ml of BrdU (31) in drinking water along with 2 mg/ml of glucose for 4, 8, and 22 days. Water bottles were shielded from light, and BrdU was replaced every 3 days. Control mice were injected with sterile PBS or were fed normal water.
Abs, staining, and flow cytometry (FCM)
The following murine mAb conjugates were used: FITC-DS-1 and
r-phycoerythrin-DS-1 (anti-IgMa (Igh-6a),
mouse IgG1b), biotin (BI)-R6-60.2 (anti-IgM, rat
IgG2a), FITC-AMS 9.1 and BI-AMS 9.1 (anti-IgDa
(Igh-5a), mouse IgG2bb), FITC-11-26c.2a
(anti-IgD, rat IgG2a, 
), purified 3F11 (anti-Bcl-2 mAb,
hamster IgG), BI-G70-204 and G94-56 (anti-hamster IgG (mixture)
mAb, mouse IgG1 and IgG2b), FITC-7G6 (anti-CD21/CD35, rat IgG2b,
, all from PharMingen, San Diego, CA), and FITC-B44 (anti-BrdU,
mouse IgG1, , Becton Dickinson, Mountain View, CA). Biotinylated Abs
were revealed by streptavidin-fluorochrome conjugates. Isotype controls
(all from PharMingen) were used where relevant.
Single cell suspensions were made from spleen and bone marrow (femur
and tibia). For surface staining, 1 x 106 cells were
reacted with 2.4G2 (anti-CD16/CD32 (Fc
III/II receptor), rat
IgG2b, , culture supernatant) before staining. Intracellular
staining to detect BrdU incorporated into DNA was performed using the
method of Tough and Sprent (35). Intracellular staining to detect Bcl-2
was performed using the method of Veis et al. (36).
FCM analysis was performed on an EPICS Elite ESP (Coulter, Hialeah, FL) flow cytometer equipped with a UV-enhanced argon ion blue laser and a helium/neon red laser. In general, negative controls were used to set voltage, and single-color positive controls were used for electronic compensation. Isotype controls were included to determine background staining. Viable cells were determined by forward and side scatter characteristics, and 1 x 104 to 5 x 104 gated events were collected. Gates in the spleen for fractions I, II, and III were set according to the method of Hardy et al. (7, 8), and the gates for fraction E and fractions I, II, and III in the bone marrow were set according to the methods of Hardy (37) and Hendriks (11). Processed samples were analyzed using EPICS Elite analysis software.
Curve fitting
For the BrdU uptake data points, curves of best fit by the least squares method were generated using MacCurveFit 1.4 (Kevin Raner Software, Mt. Waverly, Australia), a shareware program available on the World Wide Web.
Cell death analysis
Annexin V (38) and merocyanine 540 (MC 540) (39) were independently used to detect the appearance of phosphatidylserine on the outer leaflet of the cell membrane. Annexin staining was performed using standard methodology recommended by the manufacturer. Briefly, 1 x 106 splenocytes were initially incubated in 200 µl of PBS for 30 min at 37°C. This step was omitted in some experiments without any discernible alteration in the results. The cells were washed once in PBS at 4°C and then stained with phycoerythrin-IgMa and FITC-IgDa in the presence of 2.4G2 on ice. After washing once in PBS followed by another wash in annexin V binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2) the cells were reacted with 5 µl of biotin (BI)-annexin V (10 µg/ml stock; PharMingen) in 100 µl of binding buffer, gently vortexed, and incubated in the dark for 15 min at room temperature. After washing once in binding buffer at 4°C the BI reagent was revealed by incubating with SAv-Red 613 for 15 min at room temperature in the dark. The cells were washed once at 4°C, resuspended, placed on ice, and analyzed immediately.
For MC 540 staining the method of Reid et al. (39) was used. Briefly, 1 x 106 splenocytes were incubated at 37°C for 30 min. In some experiments the 37°C incubation step was omitted, and similar results were obtained. Following a wash in PBS at 4°C, 2.4G2 was added along with IgMa and IgDa fluorescent conjugates, and the cells were incubated at 4°C for 30 min. After a wash, the cells were resuspended in 200 µl of 10 µg/ml MC 540 (Molecular Probes, Eugene, OR) in PBS, incubated for 15 min in the dark at room temperature, washed once at 4°C, resuspended, placed on ice, and analyzed immediately.
To detect early apoptosis in the context of the cell cycle, combined MC 540 and Hoechst 33342 (which binds to A-T-rich regions of DNA) staining was performed using the method of Reid et al. (39) with some modifications. Briefly, 1 x 106 splenocytes were reacted with 200 µl of 5 µg/ml Hoechst 33342 (Ho 33342, Molecular Probes) in PBS and incubated in a 37°C water bath in the dark for 30 min. The cells were washed once with PBS, resuspended in 200 µl of 10 µg/ml MC 540 in PBS, and incubated for 15 min in the dark at room temperature. Following a wash in PBS, 2.4G2 was added along with FITC-IgD and BI-IgM, incubated at 4°C in the dark for 30 min, washed once, and incubated with SAv-APC for an additional 30 min at 4°C. The cells were washed once, resuspended in PBS, and analyzed immediately. Ho33342 was excited with approximately 7 mW of UV laser light (351–363 nm multiline), and fluorescence was detected with a 405-nm bandpass filter. MC 540 fluorescence was detected in the phycoerythrin channel. The gates to separate MC540 bright from dull cells were set by looking for a natural break in the staining profile of the 2n population (40).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine peripheral B cells can be subdivided into fractions I
(IgDhighIgMlow), II
(IgDhighIgMhigh), and III
(IgDlowIgMhigh; Fig. 1
) (7). We have established in separate
studies4 that normal peripheral
B cell development actually proceeds from fraction III to fraction II
to fraction I. Fraction I cells are greatly reduced in the Xid mouse
(7, 8) (Fig. 1). In Xid mice there is a 3.5-fold decrease in absolute
numbers of fraction I cells compared with normal littermates, with a
1.8-fold increase in fraction II cells and a modest 1.4-fold increase
in fraction III cells (see Fig. 1). The reduced numbers of fraction I
(IgDhighIgMlow) B cells in the absence of Btk
could reflect either the failure of maturation of cells from fraction
II to fraction I or a specific increase in the death of fraction I
cells.
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a) revealed that
while the proportions of cells undergoing apoptosis in fractions III
and II were similar in normal and Xid B cells, a twofold increase in
death in fraction I B cells was observed in the Xid mouse. Staining
with MC 540 provided similar estimates of apoptosis to those obtained
with annexin V (Fig. 2b). The twofold increase in death was
still seen when nonviable cells, defined by forward and side scatter,
were excluded from analysis (data not shown). When cells were stained
with MC 540 and Hoechst 33342, an approximately two- to threefold
increase in <2n cells was noted specifically in fraction I in the Xid
mouse (Table I). It should be noted that
the percentage of apoptotic cells in Fig. 2 and the percentage of <2n
cells in Table I differ. Phosphatidylserine exposure represents an
early marker of apoptosis, while DNA fragmentation is generally seen
later in the apoptotic process. Since phosphatidylserine-positive cells
can be phagocytosed by macrophages, many of these cells may not survive
long enough in vivo to manifest <2n changes. Regardless of the method
used, fraction I cells in the Xid mouse show a two- to threefold
increase in apoptosis. These studies indicate that a specific increase
in the apoptotic death of fraction I (but not fraction II or fraction
III cells) occurs in the Xid mouse, suggesting that the loss of these
naive end-stage B cells might account for the relative absence of
fraction I cells in the absence of Btk-derived signals.
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It has been suggested (34, 41) that Bcl-2 levels are decreased in
Xid mice and that it is the regulation of the amounts of this protein
that might account for the presumed overall proclivity of these B cells
to undergo apoptosis. However, Bcl-2 was not examined at a single cell
level in those studies, and it is possible that Btk does not actually
influence Bcl-2 expression; total Bcl-2 levels may be skewed by the
relative absence of more mature cells in Xid mice. Indeed, a direct
examination of Bcl-2 expression in fractions I, II, and III in normal
and Xid mice revealed that there is no real difference in the levels of
Bcl-2 in fractions I and II in normal and Xid B cells (Fig. 3). This suggests that the relative
absence of fraction I cells in Xid mice cannot be directly correlated
with the level of Bcl-2 expression. Further, in Xid mice, fraction III
cells can be subdivided into Bcl-2low and
Bcl-2high cells, whereas in control mice most fraction III
B cells express relatively high levels of Bcl-2 (Fig. 3). Immature B
cells in the bone marrow express low levels of Bcl-2 (42) (our
unpublished observations). The presence of increased numbers of
immature cells in fraction III of Xid mice was corroborated by our CD21
studies (Fig. 4). Newly formed cells in
the spleen are CD21dim/- (43). In fraction III cells of
Xid mice the CD21dim/- population is significantly
increased. This suggests that fraction III in Xid mice may include an
increased proportion of immature B cells.
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To obtain kinetic information on peripheral B cell maturation in the absence of Btk-derived signals, we studied cohorts of BrdU-labeled B cells in CBA/N (Xid) and CBA/Ca (control) mice. Our goal was to follow the fate of a cohort of newly generated B cells after a very brief in vivo labeling pulse of bone marrow precursors. It has been previously shown (44) that a single injection of BrdU can result in the labeling of almost all cycling cells in the bone marrow. We expected a certain proportion of activated cycling peripheral B cells to incorporate BrdU initially, but hoped to see a wave of BrdU-labeled cells develop in the periphery from bone marrow precursors. Cell cycle analyses of splenocytes from normal and Xid mice showed a very small proportion of cycling cells in normal and Xid mice (data not shown).
In the course of our BrdU labeling studies, total bone marrow and
spleen cell counts across all time points were similar to those in
uninjected controls, confirming that a single injection is nontoxic.
When bone marrow populations were examined in detail, peak BrdU
labeling was noted in IgM+IgD- cells (fraction
E, in the nomenclature of Hardy et al. (37)) at the 2 day point (Fig. 5, a and b). This
is consistent with the 1-day delay in pre-B to B cell maturation noted
in studies using [3H]thymidine as a label (45). The steep
decline in fraction E cells on day 3 was assumed to represent massive
death of B cells at the bone marrow fraction E to splenic fraction III
transition, migration to the periphery, or both. Examination of the
progression of labeled cells in the spleen revealed a marked increase
in the emergence of fraction III B cells in Xid mice (Fig. 5, c and d). There is no defect in the generation of
B cells in the Xid mouse (9, 11, 46). Our own results in Fig. 5, a and b, suggest a slight increase in the number
of newly formed B cells in the bone marrow of the Xid mouse, but this
increase is statistically not significant, representing at most a
1.2-fold increase in the peak levels of newly formed B cells in the Xid
mouse. However, there is a 3- to 4-fold increase in the total number of
BrdU-labeled splenic fraction III B cells in the Xid mouse compared
with that in CBA/Ca mice (Fig. 5, c and d). The
sharpest increase is on day 2 followed by a peak on day 3 (Fig. 5e) and a rapid decrease thereafter (data not shown).
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upper panel) the slope of BrdU-positive fraction III cells
begins to plateau soon after day 8, suggesting that a steady state is
imminent. By day 22 the steady state is evident. The slope of fraction
II cells suggests similar kinetics of cell entry and exit. The slope of
fraction I cells continued to rise on day 22, suggesting ongoing
accumulation of labeled cells in this fraction. In the Xid mouse (Fig. 6, lower panel), the initial part of the slope of
BrdU-positive fraction III cells was steep and shifted to the left
compared with that in normal mice, suggesting rapid entry of cells
within 4 days (the earliest time point studied). This is in agreement
with our earlier pulse data. The slope plateaued by day 8, supporting
an accelerated entry process. The difference between the mean absolute
numbers of BrdU-labeled fraction III cells on day 22 in Xid and normal
mice was not statistically significant (by two-tailed unpaired
Student’s t test, p = 0.1917). Given the
high rate of input of BrdU-positive cells into fraction III, one might
predict that fraction II in the Xid mouse should accumulate
BrdU-positive cells at a relatively higher rate than normal if transit
from fractions III to II was not hindered. Indeed, the slope of
fraction II in the Xid mouse continued to rise on day 22, suggesting an
ongoing accumulation of labeled cells in this fraction. The difference
between the mean absolute numbers of BrdU-positive fraction II cells on
day 22 in Xid and normal mice was statistically significant (by
two-tailed unpaired Student’s t test, p =
0.0132). These data are in line with the increase in absolute numbers
seen in fraction II of Xid mice (see Fig. 1
). Although this increase in
absolute numbers of fraction II cells can be explained by the increased
ingress from fraction III, some impairment of the maturation process
from fraction II to fraction I cannot be completely excluded. The slope
of fraction I cell accumulation in Xid mice is dramatically decreased
compared with normal, in agreement with our apoptosis data. The
difference between the mean absolute numbers of BrdU-positive fraction
I cells on day 22 in Xid and normal mice was statistically extremely
significant (by two-tailed unpaired Student’s t test,
p = 0.0001).
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suggest that in the Xid mouse there is an
increased loss of mature fraction I B cells in the periphery and
accelerated egress of newly formed B cells from the bone marrow. Taken
as a whole these results indicate that while Btk-derived signals are
essential for the maintenance of naive mature B cells (and thus for
generation of the long-lived pool), Btk is not required for the
emigration of B cells from the bone marrow to the periphery. Indeed,
accelerated emigration of newly formed B cells is noted in the Xid
mouse. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Our data, however, suggest that the loss of
fraction I cells is best explained by a specific increase in apoptotic
death in this fraction in the absence of Btk-derived signals. The
increase in newly formed cells in fraction III does not appear to
represent the mere accumulation of precursors in response to a
developmental block, but appears to reflect the accelerated export
of immature cells from the bone marrow, possibly as part of a feedback
process as discussed below.
The survival of fraction I B cells appears to be a critical maintenance
event that may be permissive for the accumulation of long-lived B
cells. Recently, Lam et al. (6) observed the failure of B cell
maintenance in the absence of Ag receptor-mediated signals in
peripheral B cells. B cell receptor-mediated signals may also be
required for the specific maintenance of fraction I B cells. This
suggests that the maturational process may be divided into
Btk-independent and Btk-dependent components (Fig. 7). The Btk-independent component of
the maturational signal presumably depends on Syk and probably
regulates bone marrow emigration and/or follicular entry (28).
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The absence of a population of IgMlow B cells was first noted in the Xid mouse >2 decades ago (47). While the relative absence of fraction I B cells in mice that lack Btk or CD45 may well be explained by the absence of a maintenance event, these results may also indirectly bear upon the relative inability of B cells to proliferate in response to Ag receptor ligation in Xid, Btk null, and CD45 null mice. Our data do not support the view that Bcl-2 levels in mature B cells are regulated by Btk signals.
It is likely that, as has long been surmised (48), immature B cells are over-represented in the periphery of Xid mice. Indeed, in more recent studies (41) the relative proportion of HSAhigh peripheral B cells has been shown to be increased in the Xid mouse, also suggesting an enhanced representation of immature cells in the periphery. There have been suggestions that Ag receptor ligation of Xid B cells may compromise the induction of survival factors or of molecules involved in cell cycle progression (49, 50, 51). It should be considered, based on our studies above, that many of these results might reflect not an intrinsic signaling defect but, rather, the examination of a skewed, less mature population of B cells in the Xid mouse. It will be necessary to re-examine some of these issues using purified B cell fractions. Increased peripheral B cell survival has been noted in Xid mice expressing a Bcl-2 transgene. However these B cells are unable to proliferate in response to Ag receptor cross-linking (52, 53). Partial rescue of peripheral B cell survival and partial correction of the defect in anti-IgM-mediated proliferation were observed in Xid B cells expressing a Bcl-XL transgene, although most "rescued" mice harbored B cells with high surface IgM and had few CD5-expressing peritoneal B cells (51). Careful analyses of the maturation of B cells and the relative proportions of B cells in fractions I, II, and III remain to be performed in Xid mice expressing either Bcl-2 or Bcl-XL transgenes.
Apart from the rapid increase in BrdU-labeled fraction III cells, we also observed a dramatic decrease in the accumulation of labeled fraction I B cells in the Xid mouse in continuous labeling experiments performed over a 22-day period. These observations are consistent with the accelerated emigration of newly formed fraction III B cells and the increased apoptotic death of fraction I B cells. Newly formed fraction III B cells represent a transient population (see Footnote 4) and probably correspond to the short-lived B cells described in other studies (29, 30, 31). Extended labeling studies examining the half-lives of individual B cell fractions in wild-type and Xid mice might lead to a more precise phenotypic definition of short-lived and long-lived B cells. In another study, when wild-type and Xid mice were allowed to ingest BrdU for 6 days, a significantly higher proportion of Xid peripheral B cells was labeled, suggesting that these cells turn over faster than wild-type B cells (54). Our results are consistent with this view, but suggest that the increased turnover in the periphery may reflect both the specific loss of potentially long-lived fraction I cells and the enhanced representation in the periphery of newly formed and presumably short-lived fraction III B cells.
In conclusion, IgDhighIgMlow peripheral B lymphocytes depend on Btk-derived signals for their maintenance. Probably as a consequence of the absence either of mature naive B cells or B-1 cells, accelerated emigration from the bone marrow of newly formed B lymphocytes ensues. Whether this accelerated emigration reflects a feedback process mediated via the B cell receptor remains to be established.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. S. Pillai, Massachusetts General Hospital Cancer Center, 149 13th St., Charlestown Navy Yard, Boston, MA 02129. E-mail address:
3 Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; BrdU, bromodeoxyuridine; FCM, flow cytometry; BI, biotin; MC 540, merocyanine 540.
4 A. Cariappa, T. J. Kim, and Shiv Pillai. Ordered events during peripheral B cell maturation. Submitted for publication.
Received for publication October 13, 1998. Accepted for publication January 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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but not in µ-only Xid transgenic mice. J. Immunol. 157:1054.[Abstract]
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