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Department of Pediatrics, Women and Infants’ Hospital-Brown University, Providence, RI 02905
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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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The restrictive nature of the cell cycle is in part regulated through an interplay between cyclins, serine/threonine cyclin-dependent protein kinases (cdks), and cyclin-dependent kinase inhibitors (CKIs) (15, 16, 17, 18). The primary cyclin/cdk complexes associated with the G1 phase of the cell cycle include cyclin D/cdk4 or 6 and cyclin E/cdk2 (19). These complexes regulate cell cycle progression from G1 into S phase by interacting with the retinoblastoma gene product (pRB), leading to its phosphorylation (20). Recent studies have suggested that there is a differential usage of D-type cyclins in various immune cell types. For example, induction of cyclins D2 and D3, but not cyclin D1, has been detected in mitogen-activated human T cells and anti-IgM + IL-4-stimulated murine B cells (13, 21). Conversely, a murine macrophage cell line has been shown to express cyclin D1 (22). In the context of human B cells, induction and overexpression of cyclins D1 and D2 have been shown to occur in mantle zone B cell tumors and in EBV-positive B cells, respectively (12, 23, 24, 25). However, it remains to be determined whether differential induction and utilization of key cell cycle regulators is associated with the B cell growth in general and with intrinsic growth characteristics of naive and GC/M B cell subpopulations in response to cytokines.
The enzymatic activity of cyclin/cdk complexes can be altered by the action of a variety of CKIs with mechanisms of action that are not yet fully understood. These inhibitors can be separated into two groups. The first group (p16, p19, etc.) acts in a specific manner by sequestering monomeric cdk4 or cdk6 to prevent their interaction with D-type cyclins (26, 27). The second group (p21, p27, etc.) is capable of inhibiting all G1 cyclin/cdk complexes (28, 29, 30). Interestingly, p27 protein levels have been shown to fluctuate throughout the cell cycle, with the highest levels occurring during growth arrest (15, 16). It is believed that a mechanism of translational or postranslational control is in part responsible for the alteration in p27 protein levels (31, 32). Moreover, it has been demonstrated that anti-IgM + IL-4 stimulation results in a rapid down-regulation of p27 in both murine and human B cells (13, 14). These findings suggest an important role for this protein in normal B lymphocyte growth in response to cytokines.
In the present study, we have utilized anti-IgM + anti-CD40-activated naive and GC/M B cell subpopulations isolated from human tonsils to demonstrate that IL-2, IL-4, and IL-10 exert their growth-promoting activities by regulating the induction and/or down-modulation of cyclin D3, cdk6, and p27. Furthermore, it is shown that naive and GC/M human B cells differ in their potential to proliferate in response to distinct stimuli by virtue of their ability or failure to regulate these cell cycle regulators.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All cell culture experiments were performed in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with L-glutamine (2 mM), penicillin (50 U/ml)/streptomycin (50 µg/ml) and 10% FCS (all from Life Technologies). Cells were seeded at a density of 1 x 106 cells/ml and incubated at 37°C under 5% CO2. B cell activation was achieved by using rabbit anti-human IgM specific for µ-chains (Dako, Carpinteria, CA), and anti-CD40 derived from hybridoma cells secreting the anti-human CD40 Ab G28.5 (American Type Culture Collection, Manassas, VA). Anti-human CD40 Ab was further purified by passing the hybridoma-conditioned medium through a protein G-Sepharose column (Bio-Rad, Hercules, CA), and the purified Ab was evaluated for its ability to activate resting human B cells to respond to IL-4 or IL-10. These preliminary experiments suggested that a concentration of 1 µg/ml was capable of stimulating significant [3H]thymidine incorporation in the presence of these cytokines. Recombinant human IL-2 and IL-4 were obtained from Genzyme, Cambridge, MA. Recombinant human IL-10 was purchased from R&D Systems, Minneapolis, MN. Rapamycin was obtained from BioMol (Plymouth Meeting, PA), and cyclosporin A was a kind gift from Sandoz (East Hanover, NJ). Baculovirus recombinant cyclin D1, D2, and D3 lysates were purchased from PharMingen (San Diego, CA). The Jurkat T cell line was obtained from the American Type Culture Collection and was routinely maintained in RPMI 1640 + 10% FCS growth medium.
Isolation of tonsillar B cells
Human tonsillar B cells were isolated as previously described (6, 33). Briefly, tonsils were obtained from routine tonsillectomy patients and finely minced, and the resulting cell suspension was depleted of T cells by rosetting with neuraminidase-treated SRBC and subsequent Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient centrifugation. Non-B cells were removed through adherence to plastic for 2 h to overnight at 37°C in a humidified atmosphere of 5% CO2. B cell populations were further purified on Percoll gradients (Pharmacia) according to density as described (34); however, a Percoll mix (2.5x PBS, 0.01 N HCl) was used in place of HBSS. Briefly, B cells were passed through a discontinuous Percoll gradient (50%, 60%, 66%, 70%). Visualization of gradients was aided by the addition of phenol red to the 50% and 66% layers. Phenotyping by FACScan (Becton Dickinson, Mountain View, CA) of cells collected at the 60% gradient (representing low density B cells enriched for IgD-CD38- memory and IgDlowCD38+ GC B cells) showed that 97% of the cells were CD19+, <28% IgD+, >61% CD38+, and >41% CD95+. Cells collected at the 70% gradient (representing high density B cells enriched for IgD+CD38- naive B cells) showed that 98% of the cells were CD19+, >75% IgD+, <16% CD38+, and <9% CD95+. Cells sedimented at the 50% Percoll gradient represented dead and apoptotic cells as determined by trypan blue exclusion and morphology. The IgD+ fraction in low density cells, which was repeatedly observed, most likely represents a IgD+CD38+ subpopulation of GC cells (35). Accordingly, combined GC/M population has been referred to as IgDlow.
Proliferation assays
Purified B cells (1 x 106 cells/ml) were activated with 1/250 dilution rabbit anti-human IgM and 1 µg/ml anti-CD40 and further stimulated with either 20 U/ml recombinant human IL-4, 100 U/ml recombinant human IL-2, or 10 ng/ml recombinant human IL-10. An aliquot of 200 µl containing 2 x 105 stimulated cells was placed in a 96-well, round-bottom microtiter plate (Falcon Labware, Oxford, CA) at times indicated and pulsed for 1 h with 1 µCi of [3H]thymidine (1 Ci/mmol; ICN, Irvine, CA). Cells were harvested, and incorporated radioactivity was measured. All assays were repeated at least three times, and all measurements were in triplicate. It is important to point out that we observed variability in response to cytokines, in particular IL-2 and IL-10, among different tonsillar preparations. For the effect of rapamycin on the growth of B cells, 1 x 105 purified unfractionated B cells were seeded in 96-well round-bottom microtiter plates in a final volume of 200 µl. Cells were grown in 10% FCS and activated with anti-IgM (1/250 dilution), anti-CD40 (1 µg/ml), and IL-4 (20 U/ml) and cultured in the presence or absence of various concentrations of rapamycin or cyclosporin A. After 72 h in culture, cells were pulsed with 1 µCi/well [3H]thymidine (1 Ci/mmol, ICN) for an additional 16 h, and incorporated radioactivity was measured.
Immunoblot analysis
Purified B cells were stimulated as described above for various time periods. Total cell extract preparation and immunodetection were performed as previously described (12). Briefly, 5 or 10 x 106 cells per time point were washed once in ice-cold PBS and harvested in 0.5 ml of lysis buffer (20 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40, 4 mM NaF, 100 µM Na3VO4, 20 mM ß-glycerophosphate, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM PMSF, 50 mM HEPES, 20 mM DTT). Protein concentration was quantified by the Bradford assay and by Coomassie blue staining of protein extracts separated on 12% SDS-PAGE. Total extract (50 µg/lane) was separated on a 12% SDS-PAGE, transferred to Hybond-enhanced chemiluminescence (ECL) nitrocellulose membranes (Amersham, Arlington Heights, IL), and blocked in TBS-T (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.2% Tween 20) containing 5% nonfat dried milk for 1 h at room temperature. Filters were then incubated overnight at 4°C with either anti-cdk2 (residues 287–298, Upstate Biotechnology, Lake Placid, NY), anti-cdk4 (Upstate Biotechnology), anti-cdk6 (clone C-21, Santa Cruz, Santa Cruz, CA), anti-cyclin D (residues 285–295, Upstate Biotechnology), anti-cyclin D2 (clone C-17, Santa Cruz), anti-cyclin D3 (clone C-16, Santa Cruz), anti-cyclin E (residues 1–128, Upstate Biotechnology), anti-p27 (clone 57, Transduction Laboratories, Lexington, KY), or anti-upstream stimulating factor (USF) (clone C-20, Santa Cruz) at a concentration of 1 µg/ml. Baculovirus recombinant cyclins D1 and D2 were used to differentiate between the different D-type cyclins. The cyclin D Ab recognizes both human cyclin D1 (36 kDa) and D2 (34 kDa). The membranes were incubated for 1 h at room temperature using appropriate horseradish peroxidase-conjugated secondary Abs and processed using the ECL detection system (Amersham).
Immunoprecipitations and in vitro kinase assays
Cell extracts were prepared as described above. Cell extracts
containing 300 µg of total protein were immunoprecipitated by
incubating with either rabbit polyclonal Ab against cdk2 (5.5 µg),
cdk4 (2 µg), cdk6 (2 µg), or cyclin D2 (2 µg) or cyclin D3 Ab (2
µg) for 1 h at 4°C with rocking. Twenty-five microliters of
protein A-Sepharose beads (Pharmacia, Milwaukee, WI) were then added,
and the samples were rocked overnight at 4°C. After centrifugation,
the pelleted beads were washed twice in lysis buffer and three times in
kinase buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM
DTT, 2.5 mM EGTA, 10 mM ß-glycerophosphate, 1 mM NaF) and suspended
in a total reaction volume of 50 µl. Phosphorylation of histone H1 or
pRB was determined by incubating the beads for 30 min at 30°C in the
presence of 1 µM cold ATP (Stratagene, La Jolla, CA), 5 µCi of
[
-32P]ATP (3000 Ci/mmol; Andotek, Irvine, CA), and 2.5
µg of histone H1 (Boehringer Mannheim, Indianapolis, IN) or 0.5 µg
of truncated retinoblastoma p56RB protein (QED Bioscience, San Diego,
CA). The reaction was terminated by boiling the samples in 2x SDS
sample buffer for 10 min. The phosphorylated substrates were then
identified by resolution in 12% SDS-PAGE followed by drying and
autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Naive, GC, and memory (M) B cells, distinguished on the basis of
IgD and CD38 expression and separated into high and low density
fractions on a Percoll gradient have been shown to exhibit differential
maturation properties (6, 10). Distinct tonsillar B cell subpopulations
were purified as described in Materials and Methods. For the
purpose of this study, naive B cells are referred to as
IgDhigh, whereas GC/M combined population is referred to as
IgDlow, since GC population consists of a IgD+
subpopulation (35). Consequently, we have used these subpopulations to
evaluate their response to cytokines in a coactivation (anti-IgM +
CD40) system. Simultaneous activation through IgM and CD40 receptor was
utilized since coactivation of B cells with anti-IgM and
anti-CD40 Abs has been shown to enhance their response to cytokines
(36). Cell aliquots were harvested at 0, 6, 18, 30, 60, 72, and 96
h after stimulation and DNA synthesis measured by
[3H]thymidine incorporation. Figure 1
A shows a typical experiment
performed with a number of tonsillar IgDhigh and
IgDlow B cell preparations stimulated in the presence of
IL-4. IL-4-dependent stimulation resulted in abundant proliferation in
both IgDhigh and IgDlow B cells, with maximal
proliferation occurring around 60 h. It appears that both cell
subpopulations enter S phase between 30 and 42 h, a finding
consistent with the kinetics of S phase entry in anti-IgM + IL-4
stimulated murine low density B cells (13). Furthermore, the high
[3H]thymidine incorporation detected at 60 to 72 h
in IgDhigh B cells and at 42 to 60 h in
IgDlow B cells indicates that a significant proportion of
these cells undergoes two or three rounds of DNA synthesis. The
extended kinetics of [3H]thymidine incorporation in
IgDhigh B cells was observed in a number of experiments,
suggesting that this B cell subpopulation may have a higher percentage
of cycling cells in response to IL-4 than IgDlow B cells.
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The results shown in Figure 1
A clearly suggest that
both IgDhigh and IgDlow subpopulations are
competent in responding to IL-4 under the activation conditions used
here. Since these subpopulations have been shown to differ in their
long term proliferating abilities in response to CD40 and IL-4 or other
cytokines (10, 37, 38), it is important to evaluate whether these
subpopulations intrinsically differ in their induction and utilization
of G1 cyclins, cdks, and CKIs. Thus, we first investigated
the expression of cyclins D1 and D2 by immunoblot analysis in
IgDhigh and IgDlow B cells stimulated in vitro
for different time periods with anti-IgM + anti-CD40 + IL-4.
Both subpopulations failed to express cyclin D1, even after treatment
for 96 h (Fig. 1B). Cyclin D2, however, was detected as
a doublet at low levels in both B cell populations (Fig. 1B). There appeared to be an up-regulation of cyclin D2 in
IgDlow B cells between 24 and 48 h after stimulation.
Total cell extract from the Jurkat T cell line was used as a positive
control because of its documented expression of cyclin D2, but not of
cyclin D1 (39). The Abs were also tested against baculovirus
recombinant cyclins D1 and D2 and were found to detect both cyclin D1
(36 kDa) and D2 (34 kDa). It is important to point out that Abs from
different vendors were used to ensure that a low level detection of
cyclins D1 and D2 was not due to the poor quality of Abs. Also, to
ensure equal protein loading, the blots were probed for the
ubiquitously expressed cellular USF. The absence of cyclin D1 and low
level of cyclin D2 expression are in agreement with the previous data
from our laboratory and others indicating that both normal,
unfractionated human B cells and EBV-negative Burkitt’s lymphoma B
cells express little or no cyclin D1 or D2 (12, 24, 25).
Although we cannot rule out the involvement of cyclin D2 in human B
cell proliferation, it is highly unlikely that this cyclin plays an
important role since it was not detected in abundance in proliferating
IgDhigh B cells (Fig. 1B). We next investigated
the presence and induction of cyclin D3 and other G1
cyclins and cdks by Western blotting using total cell extracts from
IgDhigh and IgDlow human B cells stimulated as
described above. Cyclin D3 could be detected in unstimulated B cells,
albeit at lower levels; this may be due to in vivo activation of a
fraction of these cells. Importantly, stimulation in the presence of
IL-4 resulted in further induction of cyclin D3 which peaked at 24 to
48 h (Fig. 1C). We further examined the expression of
cdk4 and cdk6, the catalytic partner(s) of D-type cyclins. cdk4 was
expressed at detectable levels but showed no significant induction in
either cell fraction during the time period examined (Fig. 1C). On the other hand, there was an increase in cdk6
expression, which was maximum around 48 h in both B cell
subpopulations (Fig. 1C). Next, the expression of cyclin E
and cdk2 was evaluated. Cyclin E induction was detectable in both
IgDhigh and IgDlow B cells and increased until
96 h after IL-4 stimulation (Fig. 1C). cdk2 expression
was significant in both subpopulations; however, it was somewhat
inducible in IgDlow B cells with little or no
change in IgDhigh B cells (Fig. 1C).
The cell cycle can be negatively regulated through the action of CKIs,
including p16, p21, or p27 (15, 16). The CKI p16 was below the level of
detection, and p21 remained unchanged at detectable but not significant
levels in both IgDhigh and IgDlow
subpopulations during the time observed (data not shown). On the other
hand, the levels of p27 were highest before IL-4 stimulation,
correlating with growth arrest, and quickly decreased by 48 h in
IgDhigh fraction and 24 h in IgDlow
fraction (Fig. 1C). The protein levels remained below
detection in IgDhigh B cells, whereas p27 began to return
to original levels by 96 h in IgDlow B cells (Fig. 1C). As a control for equal protein loading, immunoblot
analysis of USF was performed. Furthermore, the same immunoblot was
stripped and reprobed with different Abs. Therefore, the unique
regulation of p27 during IL-4-mediated B cell growth suggests an
important role played by this protein.
Cyclin D3, cdk6, and p27 regulate IL-4-mediated stimulation of growth in human B cells
Given the induction profile of cell cycle regulators in
IgDhigh and IgDlow B cells (Fig. 1, B and C), it is of importance to evaluate which
cyclin/cdk complexes are functionally active. Since cyclin D2 was seen
to be induced in at least IgDlow B cells (Fig. 1B), we investigated the ability of cyclin D2/cdk or cyclin
D3/cdk complexes to phosphorylate the pRB protein in vitro. Purified,
fractionated B cells were activated with anti-IgM, anti-CD40,
and IL-4, and total cell lysates were prepared at various time points.
Lysates were immunoprecipitated with either a polyclonal cyclin D2 Ab
or a polyclonal cyclin D3 Ab. Immunoprecipitates were then incubated in
the presence of pRB substrate, -[32P]ATP, and ATP and
kinase activity analyzed by autoradiographic analysis. The cyclin D3
immune complexes elicited a strong kinase activity which could be
detected at 12 h after IL-4 stimulation and remained constant in
both IgDhigh and IgDlow subpopulations
throughout the observed 96 h time period (Fig. 2A). In contrast, cyclin D2
immune complexes failed to mediate any significant pRB phosphorylation
in either B cell subpopulation, suggesting that cyclin D3, but not
cyclin D2, was the key D-type cyclin regulating human B cell
proliferation.
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B). To determine the
efficiency of immunoprecipitations, total cell extracts from
IgDlow B cells stimulated for 48 h with anti-IgM,
anti-CD40, and IL-4 were immunoprecipitated and probed with either
cdk4 or cdk6 Ab. cdk6 immunoprecipitated by its own Ab comigrated with
cdk6 complexed with cyclin D3 (Fig. 2B). Importantly, while
cdk4 was not part of cyclin D3-cdk complexes, it could be detected in
immunoprecipitates obtained with its own Ab.
Since the mechanism of growth inhibition by p27 involves binding to and
inhibiting the activity of the cyclin E/cdk2 complex (40) and because
p27 was somewhat differentially regulated in IgDhigh and
IgDlow populations (Fig. 1C), cyclin E/cdk2
kinase activity was evaluated in vitro in the extracts from these B
cell subpopulations. To monitor the activity of the cyclin E/cdk2
complex in IL-4-stimulated B cells, in vitro kinase assays were
performed using the histone H1 protein as a substrate. Purified
IgDhigh and IgDlow human B cells were cultured
for various times in the presence of anti-IgM + anti-CD40 +
IL-4, and cell lysates immunoprecipitated with anti-cdk2 Ab. Since
cdk2 complexes with cyclin E, it has been shown that in vitro
phosphorylation of histone H1 is due in significant part to cyclin
E/cdk2 complexes (27, 30). The kinase reactions were conducted as
described in Materials and Methods. A strong histone H1
phosphorylation activity was observed in extracts from stimulated
IgDhigh cells at 72 to 96 h (Fig. 2C),
which correlated with the elimination of p27 in these cells (Fig. 1C). However, in IgDlow B cells, maximum kinase
activity occurred at 48 h when p27 levels were lowest. These data
suggest that down-modulation of p27 is important for G1-S
transition, which correlates with active [3H]thymidine
incorporation in both B cell subpopulations (Fig. 1A) and
with functional cyclin E/cdk2 complexes. Interestingly, p27
down-modulation appears to be kinetically different between naive and
GC/M human B cells under similar activation conditions. An extended
cyclin E/cdk2 kinase activity until 96 h might explain the
proportionately higher [3H]thymidine incorporation
observed in IgDhigh B cells in response to IL-4 at 72 to
96 h (Fig. 1A).
Anti-IgM and anti-CD40-mediated activation alone results in a poor down-modulation of p27 and a weak induction of cyclin D3 and/or cdk6
Next, we evaluated the extent of B cell proliferation as well as
induction of cell cycle regulators in B cells activated with
anti-IgM + anti-CD40 alone. The data in Figure 3A revealed that anti-IgM
+ anti-CD40-mediated activation triggered a minor proliferative
response as compared with IL-4 costimulation, with maximum
[3H]thymidine incorporation (12,000 cpm for
IgDhigh; 
7500 cpm for IgDlow) occurring
between 30 and 42 h in both cell fractions.
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B). Cyclin D3 expression
was inducible at low levels initially (24 h); however, its levels
did not appear to change thereafter (Fig. 3B). Cyclin E
levels remained unchanged throughout the time examined. Interestingly,
the levels of p27 began to decrease at 48 h after activation but
remained at significant levels throughout the culture period (Fig. 3B). The presence of cyclin D3 and cdk6 until 48 h
along with a slight down-modulation of p27 could possibly account for
the minor proliferation observed in Figure 3A. On the other
hand, IgDlow B cells failed to show any induction of either
cyclin D3 or cdk6, although they could be detected at low levels (Fig. 3B). Cyclin E was expressed at significant levels but, like
IgDhigh B cells, failed to be induced. Curiously, there was
no modulation of p27 throughout the time examined, perhaps accounting
for the poor proliferation observed in this subpopulation (Fig. 3A). Since these cells begin to undergo apoptosis without
further stimulation (E. Wagner and S. Sharma, unpublished
observations), probing with USF to ensure equal protein loading was not
possible. As an alternative approach, protein concentrations were
normalized by Coomassie blue staining (data not shown). The inability
of anti-IgM + anti-CD40 treatment to induce a threshold
expression of cyclin D3 and cdk6 and to significantly decrease the
levels of p27 may explain the minor proliferation observed in these
cells. IL-10 differs from IL-4 in that it fails to stimulate a sustained expression of cyclin D3 and cdk6 in IgDhigh cells
IL-10 is another potent B cell maturation factor that exerts
varying effects depending on the origin of a cell and the presence of
other cytokines (41, 42, 43). To examine the response of
IgDhigh and IgDlow human tonsillar B cells to
IL-10, fractionated B cells were activated with anti-IgM +
anti-CD40 + IL-10 and harvested at various times; and DNA synthesis
was measured by [3H]thymidine incorporation.
IgDlow B cells displayed a robust proliferation in response
to IL-10, with maximum proliferation occurring 60 h
poststimulation (Fig. 4A). In
contrast, the addition of IL-10 to IgDhigh B cells resulted
in only minor proliferation, which was comparable to that observed in
cells activated with anti-IgM and anti-CD40, except for a shift
in the kinetics of peak proliferation (60 h vs 30–42 h). These results
indicate that IgDhigh and IgDlow human B cells
exhibit differential proliferation in response to IL-10 (42, 43).
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B).
Cyclin D3, however, was induced between 24 and 48 h, and its
levels began to decline thereafter (Fig. 4B). Curiously,
cyclin E was detectable throughout the time examined but failed to be
induced (Fig. 4B). p27, however, remained unchanged until
60 h poststimulation but decreased below detectable levels (Fig. 4B) after this time period. On the other hand, in
IgDlow B cells, cyclin D3 was induced at 24 h and
reached maximum levels between 48 and 60 h after treatment with
IL-10 (Fig. 4B). cdk6 expression was also induced at 24
h, and its levels remained constant until 96 h (Fig. 4B). Cyclin E reached maximum levels between 72 and 96
h (Fig. 4B). The p27 levels were characteristically highest
at 0 h but dropped significantly by 24 h. The original
levels, however, were restored by 96 h (Fig. 4B). The
growth of IgDlow B cells in response to IL-10 correlates
with the disappearance of p27 and the presence of cyclin D3 and cdk6
between 24 and 60 h. On the other hand, in IgDhigh B
cells, the presence of p27 up to 60 h poststimulation and the
absence of cdk6 and cyclin D3 at later time points may account for the
poor proliferation observed in Figure 4A. The varying
expression of key cell cycle regulators in IgDhigh and
IgDlow B cells is not due to unequal loading of protein,
because the USF signal was of approximately the same intensity in each
lane. Taken together, our data suggest that the differential modulation
of cyclin D3, cdk6, and p27 could possibly explain the varying
proliferative responses between IgDhigh and
IgDlow B cells in response to IL-10. IL-2 fails to significantly down-modulate p27 expression and induces a poor proliferative response in both IgDhigh and IgDlow cells
The major growth factor that stimulates proliferation and
functional differentiation of T cells is IL-2 (44). In T cells, IL-2
has been shown to induce proliferation by eliminating p27 (45). In the
context of B cells, IL-2 has been shown to act as a growth and
differentiation factor, albeit with variable effects depending on the
stage of maturation and/or activation state of the B cell (46, 47, 48). To
examine the response of IgDhigh and IgDlow
human tonsillar B cells to IL-2, anti-IgM + anti-CD40-activated
IgDhigh and IgDlow B cells were cocultured with
IL-2. Cell aliquots were taken at various times, and DNA synthesis was
measured by [3H]thymidine incorporation. In
IgDlow B cells, maximum proliferation occurred between 42
and 60 h poststimulation (Fig. 5A). However, the peak
[3H]thymidine incorporation levels were only marginally
higher (18,000 cpm) than those seen in response to anti-IgM +
anti-CD40 alone (7,500 cpm). In contrast, IgDhigh B
cells stimulated with IL-2 resulted only in a minor proliferation (Fig. 5A). Similar to treatment with IL-10, IL-2 stimulation
resulted in a shift in the kinetics of peak proliferation (60 h vs
30–42 h) as compared with anti-IgM + anti-CD40-activated
IgDhigh B cells.
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B). In IgDhigh B cells, cdk6 was induced and
reached maximum levels by 60 h; thereafter its levels began to
decline (Fig. 5B). Cyclin D3 levels increased throughout the
time of examination; peak levels were obtained between 72 and 96 h
poststimulation. p27 levels, however, remained almost constant at high
levels throughout that time. IgDlow B cells, on the other
hand, showed an inducible expression of both cdk6 and cyclin D3.
Interestingly, cyclin E was induced in both subpopulations with levels
somewhat declining at 72 and 96 h in IgDlow B cells.
p27 levels were highest at time 0 h and began to decline between
48 and 60 h. However, while p27 levels declined, they never fell
below the level of detection as observed in IL-4- and IL-10-stimulated
IgDlow B cells. Again, quantitation of USF protein
suggested an equal protein loading in each lane. Taken together, our
results suggest that IL-2 stimulation is capable of inducing the
expression of cyclin D3, cdk6, and cyclin E in both IgDhigh
and IgDlow B cells; however, it fails to efficiently
down-modulate p27, possibly explaining the poor proliferative response
in these cells (Fig. 5A). Rapamycin inhibits IL-4-mediated growth in human tonsillar B cells by restoring p27 levels
Rapamycin is a potent immunosuppressant that blocks cell growth in
the mid- to late G1 phase of the cell cycle (49). In T
cells, rapamycin blocks IL-2-mediated T cell growth by restoring p27 to
threshold levels (45). Moreover, rapamycin-mediated growth inhibition
in normal B cells or in the Burkitt’s lymphoma B cell line BJAB (50, 51) suggests its ability to act in B cells as well. Since IL-4 induced
significant proliferation in both IgDhigh and
IgDlow subpopulations of B cells (Fig. 1A) and
decreased p27 levels (Fig. 1B), we wanted to investigate
whether rapamycin would inhibit IL-4-mediated proliferation and restore
p27 levels. As a control, cyclosporin A was used in these assays.
Specifically, anti-IgM + anti-CD40 + IL-4-stimulated
unfractionated tonsillar B cells were sensitive to the
growth-inhibitory effects of rapamycin, showing >50% inhibition at
the dose of 10 ng/ml (Fig. 6A). In contrast, cyclosporin
A had no effect on IL-4-mediated B cell growth at the comparative doses
(Fig. 6A). To determine whether the rapamycin-mediated
growth inhibition in IL-4-stimulated B cells was due to an increase in
p27 levels, Western blot analysis was performed with unfractionated
tonsillar B cells treated for 20 h with various combinations of
anti-IgM, anti-CD40, and IL-4 and grown in the absence or
presence of cyclosporin A or rapamycin. Cells activated with
anti-IgM, IL-4, CD40, or anti-IgM + anti-CD40 did not
exhibit decreased levels of p27 (Fig. 6B). However, addition
of IL-4 either alone or in combination with cyclosporin A to activated
B cells (anti-IgM + anti-CD40) showed a significant decrease in
the amounts of p27 (Fig. 6B), which is indicative of
growth-promoting conditions. In contrast, addition of rapamycin
resulted in restoration of p27 back to almost original levels (Fig. 6B), which correlates with the growth inhibition observed in
Figure 6A. Taken together, these results suggest a crucial
role of p27 in regulating human B cell cycle progression in response to
growth promoting cytokines.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well documented that IL-2, IL-4, and IL-10 exhibit a redundant
characteristic in that these cytokines stimulate proliferation in
activated B cells, and the extent of the proliferative responses to
these cytokines has been shown to vary depending on the mode of
activation and the maturation stage of the B cell (5, 36, 38, 48, 52).
For example, in the reconstituted CD40 system, which depends on
coculturing B cells with CD32-transfected fibroblasts coated with
anti-CD40 mAb and cytokines, IL-4 induces proliferation in both
naive and GC/M subpopulations and supports long term, factor-dependent
growth preferentially in IgD+ B cells, whereas IL-10
induces terminal differentiation possibly in both populations (38, 42).
Curiously, IL-2 fails to exert any major activity in this system;
however, it is reasonably effective in promoting growth in B cells
activated with anti-IgM (46, 53). Corroborating these observations,
purified peripheral naive (IgD+) B cells have also been
shown to preferentially cycle in response to anti-CD40 and IL-4,
whereas memory (IgD-) B cells proliferate when stimulated
with a combination of IL-2, IL-10, and IL-3 (10). In the present study,
IL-4 was found to be a potent inducer of proliferation in both
IgDhigh and IgDlow B cells (Fig. 1A), suggesting a unique ability of this lymphokine to
stimulate both naive and GC/M B cells. IL-10 was able to induce
proliferation only in IgDlow (GC/M) B cells, but not in
IgDhigh B cells (Fig. 4A). Finally, IL-2 proved
to be a poor inducer of B cell proliferation, even in an
IgDlow B cell subpopulation (Fig. 5A). Despite
the disparity in their abilities to induce proliferation, all of these
cytokines mediated their effects through the modulation of cyclin D3,
cdk6, and p27, suggesting a possible link for their redundant activity
in the context of B cell proliferation. Together, these findings point
to intrinsic differences between naive and GC/M B cell subpopulations
in their responses to different cytokines.
Although the molecular mechanisms underlying these variances remain to
be elucidated, our results strongly suggest that active human B cell
growth is tightly controlled by cyclin D3/cdk6 complexes and the cell
cycle inhibitor p27. In the case of IgDhigh (naive) B
cells, their failure to respond to anti-IgM + anti-CD40 or to
these activators in the presence of IL-2 or IL-10 clearly correlated
with a cryptic induction of cyclin D3 and cdk6 and a poor
down-modulation of p27 (Figs. 3 to 5). Interestingly, Galibert et al.
have shown that IgD+, but not IgD-, B cells
preferentially undergo long term proliferation in response to CD40 and
IL-4 (38). In this regard, our data on the regulation of p27 protein
levels in IgDhigh B cells in response to anti-IgM +
anti-CD40 + IL-4 are of significance in that p27 remained below
detection beyond 96 h in these cells as compared with its
restoration in IgDlow B cells at this time point (Fig. 1C). Although our experiments were not conducted beyond
96 h, it is tempting to speculate that the presence of active
cyclin D3/cdk6 complexes and the absence of p27 for an extended time in
IL-4-treated IgDhigh B cells will provide long term growth
advantages.
The finding that cyclin D3, but not cyclin D2, is the key D-type cyclin
involved in human B cell cycle progression provides further evidence
for cell type-specific utilization of D-type cyclins (Fig. 2). Although
cyclin D2 was marginally expressed at least in IgDlow B
cells (Fig. 1B), it is possible that its expression was
below the threshold level required to form complexes with cdk4 or 6. In
this regard, it is important to point out that EBV has been shown to
up-regulate cyclin D2 expression in B cells (12, 24). However, it is
not yet known whether this cyclin forms functionally active complexes
with its kinase partners in EBV-positive B cells. On the other hand,
PHA-activated human T cells and anti-IgM + IL-4-treated murine B
cells have been shown to express and utilize cyclin D2 (13, 21). In
this context, our data corroborate the findings obtained with T cell
hybridomas and IL-7-stimulated precursor B cells where cyclin D3 plays
a key role in their cell cycle progression (54, 55). Despite the
evidence provided here for the unique participation by cyclin D3 and
cdk6 in human B cell cycle, cyclin E/cdk2 complex activity seemed to
pattern other cell types. For example, histone H1 phosphorylation by
cdk2 immunoprecipitates occurred when p27 protein levels were found to
be negligible (Figs. 1C and 2C). Similarly,
cyclin E was inducible and detectable in response to IL-2, IL-4, and
IL-10 under the activation conditions used here. Thus, the major
differences in cell type-specific cell cycle regulation might be
attributed to D-type cyclins and their catalytic kinase partners.
Furthermore, it is also tempting to speculate that although cdk4 and
cdk6 may both be coexpressed at significant levels, only one of them
will be selectively recruited to complex with its cyclin D partner. In
this regard, cdk4 was significantly expressed in both
IgDhigh and IgDlow B cells, but it failed to
complex with cyclin D3 (Figs. 1C and 2B). It is
possible that different cyclin D-cdk complexes would function in cells
of different origin or stage of development. For example, cdk4 is
primarily associated with cyclin D3 in precursor B cells and cdk6
complexes with both cyclin D2 and cyclin D3 in activated T cells
(54, 56).
The importance of CKIs in regulating the cell cycle has been a major
interest in both normal and malignant cells (18, 57). By virtue of its
ubiquitous presence in growth-arrested cells and elimination during
active growth, p27 has become a major focus in understanding cell cycle
progression. G1 arrest can be induced by the ectopic
expression of p27 cDNA, and there appears to be a correlation between
p27 levels and growth arrest under different mitogenic conditions (29).
Recently, it has been suggested that cell cycle progression is
dependent on the balance of sequestered p27 by cyclin D/cdk complexes
and free p27 which is able to bind and inhibit cyclin E/cdk2 complex
activity (40). In this regard, cyclic AMP- and rapamycin-induced growth
arrest has been attributed to increased levels of p27 (45, 58). In our
studies, p27 appeared to be the major player in negatively regulating
human B cell growth. Cyclin E/cdk2 kinase activity inversely
corresponded with the presence of p27 (Fig. 2C). The failure
of IL-2 to significantly down-modulate p27, particularly in
IgDhigh B cells (Fig. 5), may explain why this cytokine is
a poor inducer of B cell proliferation under the activation conditions
used in the present study. This agrees with the observations that
treatment of IL-4-stimulated human B cells with rapamycin effectively
inhibited cell growth by restoring the protein levels of p27 (Fig. 6).
These results suggest that down-modulation of p27 is necessary for
normal human B cell cycle progression and that different stimuli
exhibit varying capacities to modulate this kinase inhibitor.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Surendra Sharma, Department of Pediatrics, Women and Infants’ Hospital-Brown University, 101 Dudley Street, Providence, RI 02905. E-mail address:
3 Abbreviations used in this paper: GC, germinal center; cdk, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitor; pRB, retinoblastoma gene product; ECL, enhanced chemiluminescence; USF, upstream stimulating factor.
Received for publication June 18, 1997. Accepted for publication March 25, 1998.
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