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*
Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Majadahonda, Spain; and
Centro de Biología Molecular S.O., Consejo Superior de Investigaciones Cientificas-Universidad Autónoma, Campus de Cantoblanco, Madrid, Spain.
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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 transcription factor, B lymphocyte-induced maturation protein (Blimp-1),3 has been postulated to act as a positive regulator of B cell maturation to Ig secretion (6). The human equivalent of Blimp-1, called PRDI-BF1, is a repressor of ß-interferon gene expression and shares homologous domains with the retinoblastoma-binding RIZ protein, which was also implicated in cellular differentiation (7). Mouse Blimp-1 expression correlates with plasma cell generation in in vitro established cell lines. The transfection of either transformed mature B cell lines or anti-IgM-arrested B cells with a Blimp-1 expression vector induced the appearance of IgM-secreting cells (6, 8). We decided to elucidate the requirement for Blimp-1 expression in both T cell-independent and -dependent pathways of physiological plasma cell formation, as well as to better define the developmental stages of its action. Our results revealed a very early induction of Blimp-1 expression after LPS polyclonal B cell stimulation, clearly preceding the differentiation to Ig-secreting cells. Blimp-1 was first expressed in a population of blastic IgM+Syndecan-1(Synd)+CD43+ preplasma cells. Both the emergence of the later cell population and differentiation to IgM-secreting plasma cells were suppressed by Blimp-1-specific antisense oligonucleotides (ON). In contrast, plasma cell differentiation and IgM/IgG1 class switching secondary to Th cell stimuli (Th2 cell line, anti-CD40 + IL-4) were neither accompanied by Blimp-1 up-regulation nor affected by anti-Blimp-1 ON treatments.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and C3H/HeJ mice, 8–12 wk old, were maintained in the specific pathogen-free animal facilities of the Instituto de Salud Carlos III. Total splenocytes and purified B cells (see below) were cultured at 106 cells/ml in RPMI 1640 medium (Life Technologies, Paisley, U.K.) supplemented with 10% FCS (Labtech, Uckfield, U.K.), 2 mM L-glutamine, 1 mM pyruvate, 50 mM 2-ME, 10 mM HEPES, and antibiotics. Cultures were maintained at 37°C and 5% CO2 in a humidified atmosphere. Escherichia coli LPS (Difco, Detroit, MI) was added to 25 µg/ml. The SR.D10 cell line is an autoreactive variant of the CD4+ Th2 cell line D10.G4.1, known to secrete IL-4, IL-6, and IL-10 (9, 10), and it was maintained in vitro by coculture with mitomycin C-treated, syngeneic C3H/HeJ feeder splenocytes in Click’s medium supplemented with 10% FCS and 100 µg/ml conalbumin (Sigma Chemicals, St. Louis, MO). For Th cell assays, SR.D10 cells were treated with mitomycin C and cocultured with C3H/HeJ-purified spleen B cells (cell ratio of 1 SR.D10 vs 100 splenic B cells). Cultures with anti-CD40 FGK45.5 mAb (10 µg/ml) and mouse rIL-4 (100 U/ml) were set up as described before (11). Cell proliferation was measured after a 12-h pulse of 1 µCi [3H]thymidine, and incorporated radioactivity was quantified by scintillation counting (Wallac LKB, Turku, Finland).
Flow cytometry analysis and sorting
Fluoresceinated anti-leukosialin S7 (anti-CD43) (12) and biotinylated anti-Synd-1 (13) mAb were purchased from PharMingen (San Diego, CA). Anti-IgM (331.12) (14) mAb was purified from culture supernatants by affinity chromatography on protein G columns (Pharmacia, Uppsala, Sweden) and fluoresceinated by standard methods. Phycoerythrin-conjugated Streptavidin was from Southern Biotechnology Associates (Birmingham, AL). Two-color stainings were performed as previously described (15). Dead cells were excluded on the basis of forward- and side-light scatter parameters, and propidium iodide staining. Specific mAb signals were defined against the fluorescence provided by isotype-matched, irrelevant mAb. Flow cytometry analyses were performed on an EPICS-XL flow cytometer (Coulter Electronics, Hialeah, FL). Cell sorting of splenic IgM+ B cells and cultured IgM+Synd+ cells were undertaken in a FACStar (Becton Dickinson, Mountain View, CA), and reanalyses of the purified cell samples were done in the EPICS-XL analyzer, to control the degree of purity. The frequencies of contaminating cells in purified samples were <5% after flow cytometry reanalysis.
ELISPOT assays
Ninety-six-well ELISA plates (Nunc, Kamstrup, Denmark) were
precoated with either goat anti-mouse total Ig or rat
anti-mouse IgG1 mAb (Southern Biotechnology Associates) for
detection of IgM- and IgG1-secreting cells, respectively (3 µg/ml),
and blocked with 1% gelatin/PBS. Cells were removed from cultures,
washed three times, and counted. Serially diluted cells were seeded in
triplicate in 1% FCS/RPMI 1640 and incubated at 37°C overnight in
the coated plates. After washing, IgM- and IgG1-secreting cells were
detected by incubation with biotinylated goat anti-mouse IgM
(Southern Biotechnology Associates) or rat anti-mouse 
-chain mAb
(187.1) (16), respectively. The plates were washed again
and incubated with Streptavidin-conjugated alkaline phosphatase
(Southern Biotechnology Associates). Plates were developed by adding
5-bromo-4-chloro-3-indolyl-phosphate (Sigma) in alkaline buffer
containing 1% low melt agarose (Sigma) and incubated at 37°C.
Developed spots were counted under a dissecting microscope.
RT-PCR analyses
Total RNA was isolated and cDNA was prepared as described (17), using 1 µg oligo(dT) as primer and avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). Equal amounts of cDNA were used to amplify Blimp-1, BSAP, and ß-actin transcripts. Enzymatic amplifications were performed as follows: initial denaturation (95°C, 5 min), incubation (80°C, 5 min), addition of 2.5 U of Taq DNA polymerase (DNAzyme, Espoo, Finland), and then 30 cycles of the following steps: denaturation (95°C, 1 min), annealing (60°C, 1 min), and elongation (72°C, 1 min). The final polymerization step was extended for 10 min more. Primers for ß-actin are described (15); for Blimp-1 and BSAP amplifications, the following primers were designed: Blimp-1 sense: 5'-TCAGCCTCTTCCCTAGGTTGTATC-3'; Blimp-1 reverse: 5'-AATCTTAAGGATCCATCGGTTCAAC-3'; BSAP sense: 5'-CAGCATAGTGTCTACAGGCT-3'; BSAP reverse: 5'-GTCTCGGCCTGTGAAATAGG-3'.
The amplification product (one-fifth of each) was separated electrophoretically on 1.5% agarose gels and then transferred to Zeta-probe membranes (Bio-Rad, Hercules, CA) after treatment with 0.4 M NaOH. Hybridization was performed with ß-actin and Blimp-1 probes, 32P-labeled by random priming (18). The ß-actin insert was the PstI fragment of the plasmid p41AL (19). The Blimp-1 insert was obtained after cloning the 1178-bp fragment of the PCR-amplified Blimp-1 from LPS-activated B cells into the Bluescript vector (Stratagene, La Jolla, CA). The following internal ON was used as a 32P-labeled BSAP probe: 5'-AAGCCATGGCTGAATACTCTG-3'. PCR signal intensities were quantified by densitometry (Fujibas-1000 detector, Fuji, Tokyo, Japan). Control experiments of titration were performed in order to ensure the quantification of the cDNA samples: aliquots of the amplifications were removed at 20, 25, 30, or 35 cycles in preliminary PCRs, and serial dilutions of the positive cDNA samples were PCR amplified for 35 cycles.
In vitro protocols with phosphorothioate-modified ON
Phosphorothioate ON were prepared by Isogen Bioscience BV (Maarssen, The Netherlands). These consisted of three 18-mer that were antisense (AS), sense (S), and non-sense (NS) for the DNA sequence spanning the fragment of the murine Blimp-1 cDNA located -4 to +14 from the first ATG: AS: 5'-TAAGCCTCTCTCATGTCC-3'; S: 5'-GGACATGAGAGAGGCTTA-3'; NS: 5'-ARSRWWRWSRWRRWRWWA-3' (W: A + T; R: A + G; S: G + C).
Spleen B cells were purified by cell sorting, washed twice with BSS, and then cultured in serum-free Opti-MEM (Life Technologies) supplemented with 5% NCTC 109 medium (BioWhittaker, Walkersville, MD) in basal conditions, under stimulation with LPS, anti-CD40 mAb + IL-4, or cocultured with SR.D10 cells, as described above. Different concentrations of ON were added at the start of cultures and for various lengths of time.
Western blot analysis
Cell extracts (107 cells/sample) were obtained after lysis in: 20 mM Tris-HCl (pH 7.5), 20% glycerol, 100 mM KCl, 2 mM DTT, and 1 mM PMSF. The resulting samples were boiled for 5 min in 2x SDS Laemmli sample buffer (0.04 M Tris-HCl (pH 6.8), 2% SDS, 5% ß-mercaptoethanol, 10% glycerol, 0.002% bromphenol blue), electrophoresed in SDS-PAGE (6%), and then transferred to Hybond ECL nitrocellulose membranes (Amersham Life Science, Little Chalfont, U.K.). The total amount of protein/sample was determined before loading, using a protein determination reaction (Bio-Rad). As a further loading control, the lower part of the gel was sliced and stained with Coomasie blue, and two selected bands/lane were quantified (Gelworks 1-D intermediate program, UVP, Cambridge, U.K.). Filters were blocked with 5% fat-free dry milk in PBS/0.05% Tween-20 and incubated overnight with anti-Blimp-1 rabbit polyclonal antiserum (generously provided by Dr. M. M. Davis, see 6), washed, and further incubated with HRP-conjugated anti-rabbit Ig (Southern Biotechnology Associates; 2 h at room temperature). Filters were developed using ECL Western blotting detection reagent (Amersham).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although it is absent in pre-B/B cell lines, Blimp-1 expression is
detected in tumors with plasma cell phenotype and in plasma
cell-containing cultures (6). We have dissected the
kinetics of Blimp-1 transcription in normal splenocytes activated with
LPS mitogen. The appearance of plasma cell markers in flow cytometry,
Ig-secreting cells in ELISPOT assays, and Blimp-1 transcripts in
specific RT-PCR were analyzed in parallel. Blimp-1 was well expressed
after a few hours of LPS culture, long before significant numbers of
IgM-secreting cells were detected in the same cultures (24 to 48
h) (Fig. 1
A, white triangles). Blimp-1 specific
transcripts reached half of their maximum values (obtained in 72-h cell
cultures) at 18–24 h, when only small numbers of IgM-plasma cells
(relative to those of IgM-secreting cells obtained at 72 h) had
developed. The mean ratio between specific Blimp-1 gene
expression and IgM plasma cells subsequently peaked at early time
points (18 h) and later decreased, although IgM plasma cells were still
expanding in the cultures (Fig. 1A, black circles).
These data show that the Blimp-1 gene is up-regulated very
early after LPS B cell stimulation, and probably at cellular stages
previous to fully mature plasma cells.
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B) and progression through the rest of
the cell cycle. A fraction of these activated B cells progressively
acquired features of differentiating plasma cells. We analyzed in the
LPS stimulated cultures the expression of CD43 and Synd-1 Ags, which
are normally present in bone marrow pre-B cells, down-regulated in
mature B cells, and reexpressed again in IgM-secreting plasma cells
(12, 20). A novel population of blastic
IgM+Synd+CD43+ cells emerged very
early in the cultures (2% in 24-h cultures) and expanded during the
following days (5% and 10% at 48 and 72 h, respectively) (Fig. 1B, and data not shown). The
IgM+Synd+ cell population contained most of the
IgM-secreting cells of the cultures (as observed after its purification
with FACS sorting and IgM-specific ELISPOT analyses; data not shown),
although it also included other preplasma cell stages. The frequencies
of IgM+Synd+CD43+ cells in the LPS
cultures were higher (10-fold) than the relative number of
IgM-secreting cells present in the same cultures. The early Blimp-1
transcription shown above (Fig. 1A) suggested that this gene
might be selectively up-regulated at the level of the
IgM+Synd+CD43+ B cell/plasma cell
transitional stage. We tested this possibility directly by analyzing
the Blimp-1 transcripts present in IgM+Synd+
cells purified by FACS sorting, in quantitative RT-PCR analyses. The
relative expression of Blimp-1 (compared with the one of ß-actin) was
10–20 times higher in the purified IgM+Synd+
cells than in total LPS cultures (Fig. 1B). Blimp-1
transcripts were barely detected in the sorted Synd-
population of the same cultures (data not shown). In contrast,
transcription of the BSAP gene, expressed in mature B cells
(5, 21), was strongly down-regulated in the LPS cultures
and in the purified IgM+Synd+ cells (Fig. 1B). Inhibition of Blimp-1 protein expression suppresses the generation of IgM+Synd+ preplasma and IgM-secreting plasma cells in LPS-activated spleen B lymphocytes
Blimp-1 expression was previously shown to be sufficient for
plasma cell maturation, as demonstrated by Blimp-1 transfection into
either nonsecreting B cell lines or anti-IgM-blocked B cells
(6, 8). We asked whether Blimp-1 activity was also
necessarily required for plasma cell differentiation.
Phosphorothioate-modified, Blimp-1-specific S, AS, and NS ON were
generated and added to LPS-stimulated cultures of FACS-sorted, C3H/HeJ
spleen B cells. To reduce sequence-independent effects of ON, we
included no CpG motifs (B cell stimulatory) in their sequences, and we
worked with low ON concentrations (1–10 µM) (22, 23).
These experiments revealed a specific, dose-dependent reduction of the
IgM+Synd+ preplasma cell population in cultures
treated with AS Blimp-1 ON (Fig. 2A). More important, the
emergence of IgM-secreting plasma cells was progressively reduced in a
time- and dose-dependent manner with the AS Blimp-1 therapy. Thus,
IgM-secreting cells of AS Blimp-1 ON-treated cultures (3 µM) reached
only one-third to one-fourth of those present in 3-day basal LPS
cultures. Some nonspecific reductions in plasma cells were observed
with both S and NS Blimp-1 ON, but they were significantly different
from those induced by Blimp-1 AS ON (p < 0.05,
for all the ON doses in 72-h cell cultures, after applying the
two-tailed unpaired Student t test) (Fig. 2B).
Cell cultures extended until 96 h with 3 and 10 µM doses of ON
provided similar results, although nonspecific cell death increased
(data not shown). In order to check for the cellular level of action
and the specificity of the anti-Blimp-1 ON treatment, we analyzed
both Blimp-1 gene transcripts by RT-PCR and Blimp-1 protein
levels by means of Western blots with a Blimp-1 antiserum
(6), in selected samples of the cell cultures. While
Blimp-1 gene transcription remained unmodified with/without
ON treatments (Fig. 2C), the small Blimp-1 protein levels
detected in ex vivo B cells were augmented upon LPS stimulation
(3-fold) and this increase was suppressed with AS ON, but not with S ON
(Fig. 2D). Blimp-1 AS ON thus acted at the
posttranscriptional level of expression. We here conclude that
Blimp-1 gene activity is necessary for normal spleen B
lymphocyte differentiation to IgM-secreting plasma cells in T
cell-independent conditions. The Blimp-1 role is first observed at the
level of the generation of IgM+Synd+ preplasma
cells.
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T-B cell collaboration is crucial to efficient Ab responses to
Ags, in order to trigger the processes of Ig switching and somatic
hypermutation in the germinal center reaction. This intercellular
cooperation is sustained by defined receptor-ligand pairs (e.g.,
CD40-CD40L, OX40-OX40L) and T cell-derived soluble factors (24, 25). Since LPS mitogen directly activates B cells in the absence
of T cell help, we decided to study whether Blimp-1 transcription
factor was also required in bona fide T cell-driven plasma cell
formation. We cocultured spleen B cells from C3H/HeJ mice
(H-2k) with syngeneic autoreactive SR.D10 Th2 cells
(9, 10). We then analyzed B cell proliferation and the
production of IgG1-secreting cells in different basal conditions
(medium alone, LPS, SR.D10 T/B cell coculture) and following the
addition of S and AS Blimp-1 ON. B cell proliferation (as defined by
[3H]thymidine incorporation) was unmodified by ON
treatments under any condition tested (Fig. 3; top columns). The
production of IgG1-secreting cells was significantly diminished upon
the addition of AS Blimp-1 ON to the LPS cultures (Fig. 3; bottom
left columns), as it also happened for IgM-secreting cells (see
Fig. 2). In contrast, no change in IgG1 plasma cell numbers was
observed in the T/B cell cocultures, in the presence of either S or AS
Blimp-1 ON (Fig. 3; bottom right columns). Similar negative
results were observed for the IgM-secreting cells (not shown). Blimp-1
transcripts were not induced in these T/B cultures (2.8-fold vs
146-fold of day 0 specific Blimp-1 signals detected in 96-h
SR.D10/C3H/HeJ B cell and LPS cultures, respectively; data not shown
from three independent experiments). The findings described here
suggest 1) that B cell proliferation rates are independent of Blimp-1
activity, and 2) that Blimp-1 may also be dispensable for T cell-driven
plasma cell differentiation and Ig switching.
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Anti-CD40 mAb and IL-4 cooperate in the differentiation of B cells
to IgG1 plasma cells (25, 26, 27), and partially mimic
conventional Th activities. Both stimuli together drive the B cells
away from early IgM secretion and commit them to isotype switching and,
thus, presumably to enter the long-lived plasma and memory cell pools
(28). We set up cultures of spleen B cells stimulated with
anti-CD40 mAb and IL-4 as a surrogate of T cell help
(11), in the presence or not of Blimp-1 ON treatments. A
population of IgG1-secreting cells newly appeared in the
anti-CD40 + IL-4 cell cultures. Neither the
IgM+Synd+CD43+ cell subset nor a
significant up-regulation of Blimp-1 gene transcription was
observed, when compared with parallel LPS cell cultures (Fig. 4, A and B). While
the numbers of IgG1-secreting cells did not change upon Blimp-1 AS ON
therapy in anti-CD40 + IL-4 cell cultures, they were
selectively decreased in LPS cell cultures submitted to the same
Blimp-1 AS ON (Fig. 4C). Cell cultures stimulated with
either anti-CD40 mAb alone or with IL-4 alone did not show either
the IgM+Synd+CD43+ cells, the
Blimp-1 up-regulation, or IgG1-secreting plasma cells. A consistent
decrease of basal Blimp-1 transcription levels was observed in the IL-4
enriched cell cultures, as has been described (29). BSAP
transcripts of mature B cells were decreased in all the stimulated B
cell cultures (IL-4, anti-CD40 mAb, anti-CD40 mAb + IL-4,
and LPS), even in those cases in which Blimp-1 was not up-regulated
(Fig. 4). We conclude that B cell differentiation to IgG1-secreting
plasma cells secondary to CD40/CD40L + IL-4 signals follows a
distinctive pathway in the absence of
IgM+Synd+CD43+ preplasma cells, and
without the requirement of Blimp-1 gene expression and
activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B cell activation through the OX40/OX40L pathway, which is up-regulated by CD40 stimuli, also gave rise to Ig-secreting cells in the absence of Blimp-1 expression (30). Persistent stimulation of the CH12 B cell line with CD40L-transfected cell lines induced a down-regulation of Blimp-1 together with an arrest of B lymphocyte terminal differentiation (29). These later data do not disagree with ours because in physiological conditions activated T cells rapidly down-regulate CD40L (31, 32). Also, T cell-derived IL-4 concurs with CD40 in B cell differentiation to IgG1 plasma cells (26), while being unable to up-regulate Blimp-1 transcription (8, 29). The expression of Synd-1, normally controlled by Blimp-1 (6), was also shown to be absent during Th2-driven differentiation to IgG1+ cells (33). It has been described that cytokines can modulate Blimp-1 transcription (8). In particular, IL-6 is a major inducer of plasma cell differentiation (34) and up-regulates Blimp-1 transcription in the CH12 cell line (29). In recent experiments, we found small IL-6 transcript levels in LPS-stimulated B cells (not shown), as described for CH12 cells (34). The addition of IL-6 to the B cell cultures only provoked modest (3-fold) increases in Blimp-1 transcription. Moreover, LPS-activated B cells from IL-6-/- and wild-type mice (35) showed similar Blimp-1 transcription (our manuscript in preparation). Although we cannot completely exclude that IL-6 (and/or other cytokines) may contribute to the up-regulation of Blimp-1 by LPS, IL-6 is not a necessary requirement.
Other transcription factors are important at different levels of B cell
activation/proliferation. Oct-2 and the OCA-B coactivator act on early
B cell activation (36, 37). The different complexes of the
NF-B/Rel family of transcription factors play relevant roles during
B cell proliferation and Ig switching (3). BSAP and
c-myc regulate the growth rates of mature B cells
(5), the later one being suppressed by Blimp-1
(38). c-myc down-regulation may be responsible
for the apoptosis-inducing activity of Blimp-1 in selected cell lines
(39). Our data show that BSAP down-regulation during T
cell-dependent plasma cell differentiation (40) is not
mediated by Blimp-1 activity.
Based on the above findings, it is tempting to speculate that
distinctive molecular requirements are involved in the production of
different types of plasma cells. A provisional scenario of plasma cell
differentiation could be delineated (Fig. 5): Ag stimulation of naive B lymphocytes
drives them to activation, proliferation (c-myc and BSAP
dependent), clonal expansion, and a Blimp-1-controlled checkpoint
of apoptosis/differentiation to plasma cells (39). The
later selective step may occur at the level of
IgM+Synd+CD43+ preplasma cells.
Surviving cells will give rise to the early wave of IgM-secreting
plasma cells of the primary response. T cell dependent, Ag-primed B
cells receive signals for proliferation/survival from activated T cells
(CD40L expressing) (41, 42), which bias them away from
early death/plasma cell differentiation toward Ig-switched plasma cells
(28). The expression of BSAP and the absence of Blimp-1
transcription factors (40) might be, among others,
molecular characteristics of the T cell-dependent pathway of plasma
cell differentiation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. María-Luisa Gaspar, Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, E-28220 Majadahonda, Madrid, Spain. E-mail address:
3 Abbreviations used in this paper: Blimp-1, B lymphocyte-induced maturation protein; Synd, Syndecan-1; ON, oligonucleotides; AS, antisense; S, sense; NS, non-sense; ELISPOT, enzyme-linked immunospot; L, ligand.
Received for publication August 31, 1998. Accepted for publication April 26, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T-cell receptor (TCR): regulation of TCR-induced activation. Embo J. 13:90.[Medline]
heavy chain class switching. Immunity 5:319.[Medline]
-1 and 
Ig gene expression in murine B cells: IL-4 and the CD40L-CD40 interaction provide distinct but synergistic signals. J. Immunol. 155:5637.[Abstract]
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C. Angelin-Duclos, G. Cattoretti, K.-I Lin, and K. Calame Commitment of B Lymphocytes to a Plasma Cell Fate Is Associated with Blimp-1 Expression In Vivo J. Immunol., November 15, 2000; 165(10): 5462 - 5471. [Abstract] [Full Text] [PDF]
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) and IgM-secreting
cells (
) were calculated in simultaneous RT-PCR and ELISPOT assays,
respectively (left scale of ordinates). The data are
expressed as relative values of the maximum obtained at 72 h of
culture. RT-PCR signals obtained for Blimp-1 and
ß-actin genes were quantified by densitometric analyses.
The Blimp-1 message levels were defined for each time point as follows:
100 x (OD Blimp-1/OD ß-actin)/(maximal OD of Blimp-1/maximal OD
of ß-actin). A total of 107,000 ± 16,000 IgM-secreting
cells/106 cultured cells were detected in 72-h LPS
cultures. The ratios between Blimp-1 transcription levels and
IgM-secreting cells were calculated (•; right scale of
ordinates). The data obtained in five independent experiments are
shown in the figure (mean ± SD). B, Two-color
stainings of LPS-stimulated BALB/c splenocytes were performed at the
starting point and in 72-h cultures by using fluoresceinated
anti-CD43 and anti-IgM mAb, and biotinylated anti-Synd mAb,
as described in Materials and Methods. Cell size (as defined
by forward scatter) is shown in histograms for normal splenocyte
(left histogram), and for 72 h LPS
IgM+Synd- and
IgM+Synd+ cell populations (right
histograms: shaded and empty areas, respectively). All data are
displayed in log10 fluorescence scales, except for forward
light scatter (FSC), which is linear. In the bottom, RT-PCR assays for
Blimp-1, BSAP, and ß-actin are represented. They were performed on
normal splenocytes, 72-h LPS spleen cell cultures, and 72-h LPS
IgM+Synd+ cells purified by FACS sorting. The
Blimp-1/ß-actin message ratios obtained in the representative
experiment shown were 0.31 for normal splenocytes, 26 for 72-h LPS
spleen cultures, and 337 for 72-h IgM+Synd+
cells.
