PHARMACOLOGY AND CELL METABOLISM
Alcohol Affects the Late Differentiation of Progenitor B Cells
HaoWang*, Huijuan Zhou, Simon Mahler, Robert Chervenak and Michael Wolcott
Department of Microbiology and Immunology, Louisiana State University Health Science Center at Shreveport, 1501 Kings Highway, Shreveport,
LA 71130, USA
*Corresponding author: Department of Emergency Medicine, JPS Health Network, 1500 S. Main Street, Fort Worth, TX 76104, USA.
(Received 13 June 2010; in revised form 14 October 2010; accepted 15 October 2010)
Abstract — Aims: Previous studies show that alcohol exposure can affect the differentiation of progenitor B cells. Before final com-
mitment to a B lineage, progenitor B cells usually undergo several important stages. However, it is still unclear whether alcohol
alters B cell differentiation at which stages. The aim of this study was to determine which stage(s) of progenitor cell differentiation
are affected by alcohol and to elucidate the mechanism(s) responsible for the effect of alcohol on B cell differentiation. Methods:
Oligoclonal-neonatal-progenitor (ONP) cells from bone marrow cells of 2-week-old mice were cultured under different conditions
in vitro with or without the exposure of 100 mM alcohol. Phenotype analysis was performed at different time points and expression
levels of transcription factors (TFs) and cytokine receptors were measured quantitatively and kinetically. Results: After 3 days
in vitro culture, ONP cells differentiated into two populations: B220−CD11b−and B220−CD11b+cells. B220−CD11b−cells can
further differentiate into B lineage cells only with the support of B220−CD11b+cells. Cells exposed to 100 mM of alcohol during
the first 3 days of culture showed no statistically significant difference in B cell formation after 12 days compared with the control
group. However, cells exposed to alcohol from Day 4 till the end of culture yield very few B cells. Expression levels of TFs and
cytokine receptors were down-regulated kinetically among ONP cells co-cultured with the addition of 100 mM alcohol.
Conclusions: Alcohol affects the ONP cell differentiation into B lineage at a late stage. Alcohol also down-regulates the expression
level of TFs and cytokine receptors resulting in the impairment of B cell differentiation.
Alcohol abuse has a tremendous impact on human health and
(Anonymous, 2000; Blot, 1992; Doll et al., 1994; Driver and
Swann, 1987; Fuchs et al., 1995; MacGregor and Louria,
1997; Ringborg, 1998; Szabo, 1999). Chronic alcohol abuse
adversely affects the homeostasis of the hematopoietic system
causing deregulation of many of the developmental pathways
and in some cases lead to cytopenia (Chanarin, 1979, 1982;
Cowan, 1980; Dai et al., 2000; Hillman, 1975; Michot and
Gut, 1987; Seppa et al., 1991). The deleterious effects of
alcohol abuse on hematopoiesis are apparent in both periph-
eral blood and bone marrow (BM). Chronic abuse of alcohol
also leads to alterations in immune regulation that can be man-
ifested as immunodeficiency or autoimmunity (Cook, 1998),
with the consequence of increased susceptibility to disease
and damage to organ systems, respectively.
The immunologic effects of alcohol abuse tend to be long
lived and are not readily reversible by short periods of absti-
nence. There are many clinical and experimental studies that
support the hypothesis that alcohol abuse leads to immuno-
deficiency in both the innate and adaptive immune systems
(Baker and Jerrells, 1993; Cook, 1998; MacGregor, 1986;
MacGregor and Louria, 1997; Szabo, 1999). Alcohol can
result in the loss of lymphocytes from both primary and sec-
ondary lymphoid organs (Sibley et al., 1995). There is a
growing awareness, from studies of alcoholic patients, that
the regulation of B lymphocytes is particularly sensitive to
chronic alcohol exposure (Cook, 1998). The capacity of the
bone marrow to supply B lymphocytes far exceeds the
number of mature B cells transported to the periphery
(Forster and Rajewsky, 1990; Opstelten and Osmond, 1983).
Furthermore, suppression of bone marrow B-cell develop-
ment may not always be immediately apparent in the
periphery. For example, in pregnant animals, the increased
estrogen causes >90% decline in bone marrow B-cell precur-
sors with no apparent decline in the number of mature per-
ipheral B lymphocytes (Kincade et al., 1994; Kouro et al.,
2001; Medina and Kincade, 1994; Medina et al., 2000).
Therefore, a possible explanation, among others, for the B
lymphopenia observed in alcoholics is, over a period of time
alcohol could affect bone marrow B-cell development to an
extent that is, eventually, manifested as a decrease in the
number of peripheral B lymphocytes.
Recent work from this laboratory has shown that fetal
alcohol exposure can affect bone marrow B-cell development
(Biber et al., 1998; Moscatello et al., 1999; Reimold et al.,
1996; Wolcott et al., 1995). In these studies, we showed that
fetal exposure to alcohol impeded neonatal B-cell develop-
ment in both the spleen and the bone marrow through the
first 5–6 weeks of life. These studies have led to the identifi-
cationofa progenitor cell,
progenitor (ONP), which has the capacity in in vitro cultures
to differentiate to both B lymphocytes and myeloid linage
cells depending on the growth conditions and cytokines
present. When ONP cells are isolated from neonates that
were exposed to alcohol in utero, they demonstrate a greatly
reduced capacity to respond to interleukin (IL)-7 and commit
to the B lineage but have no diminution in the response to
the granulocyte–monocyte colony-stimulating factor (GM-
CSF) growth factor and commit to the myeloid lineage.
Recent studies also showed that ONP cells isolated from
normal neonatal mice and cultured in vitro in the presence of
alcohol also failed to respond to IL-7 and commit to the B
lineage. The results of these studies confirmed that alcohol
affected the cell fate decisions of this progenitor cell to
commit to the B lineage but not to the myeloid lineage.
Hierarchical expression of transcription factors (TFs) and
growth factor receptors serve as important developmental
Alcohol and Alcoholism Vol. 46, No. 1, pp. 26–32, 2011
Advance Access Publication 22 November 2010
© The Author 2010. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved.
expression of the TFs PU.1, early B-cell factor (EBF) and
the B cell regulator protein (Pax5) and signaling through
growth factor receptors, including the tyrosine kinase Flk2/
Flt3 and IL-7R, are important steps in the differentiation of
progenitor cells to the B lineage (Adams et al., 1992;
Busslinger, 2004; DeKoter et al., 2002; Greenbaum and
Zhuang, 2002; Medina and Singh, 2005). TFs activate or
repress target genes and signaling receptors induce or modify
the activities of gene regulatory proteins. Previous studies in
alcohol-exposed animals failed to up-regulate IL-7Rα and
had decreased expression levels of down-stream EBF and
Pax5 during in vitro culture under conditions that favored
differentiation to the B lineage (Wang et al., 2009). This
indicated the impaired commitment of progenitors to the B
Despite evidence that alcohol affects B-cell differentiation
from an ONP, it is unclear whether it alters B-cell develop-
ment at an early stage, late stage or in both stages The aim
of this study was to determine which stage or stages of pro-
genitor cell differentiation are affected by alcohol and to elu-
cidate the mechanism(s) responsible for the effect of alcohol
on B-cell differentiation.
ONP cellssorted from
MATERIAL AND METHODS
Six- to 8-week-old male and female C57BL/6J mice were
purchased from the Jackson Laboratories (Bar Harbor, ME,
USA) and allowed to acclimate for 1 week prior to exper-
imental use. Mice were bred in house and 2-week-old neo-
natal mice were used in all experimental protocols. All
procedures utilizing animals have been reviewed and
approved by the Animal Care and Use Committee (Protocol
The following monoclonal antibodies (mAbs) were purchased
from either eBioScience(San
BD-PharMingen (San Diego, CA, USA): Allophycocyanin
(APC)-conjugated anti-B220, Phycoerythrin (PE)-conjugated
anti-B220 (Clone RA3-6B2), APC-conjugated anti-CD19
(Clone 6D5), APC-conjugated anti-Gr-1 (Clone RB6-8C5),
APC-conjugated anti-CD3 (Clone 17A2), APC-conjugated
53-6.7), APC-conjugated and FITC-conjugated anti-CD11b
(Mac-1, Clone M1/70), APC-conjugated anti-Ter-119 (clone
Ter-119), APC-conjugated anti-Sca-1 (Ly-6A/E, Clone D7),
APC-Cy7-conjugated anti-CD117 (c-Kit, Clone 2B8) and
FITC-conjugated anti-CD24 (HSA, clone M1/69). Two
isotype controlswere purchased
FITC-conjugated Rat IgG2b κ, control for rat IgG2b anti-
bodies and PE-conjugated Rat IgG2a κ, control for rat IgG2a
antibodies. Two other isotype controls, APC-Cy7-conjugated
Rat IgG2a κ and APC-conjugated Rat IgG 2b κ, were pur-
chased from CalTag (Burlingame, CA, USA). PE-conjugated
anti-CD43 (Clone S7) and APC-Cy7-conjugated anti-CD19
(Clone 1D3) were purchased from PharMingen (San Diego,
CA, USA). Anti-Fc receptor mAb [anti-crystallizable frag-
ment receptor (FcR), clone 2.4G2] was produced in house
from cells purchased from American Type Culture Collection
(Rockville, MD, USA).
Recombinant mouse stem cell factor (SCF), mouse Flt-3/
Flk-2 ligand (FL), mouse IL-3, mouse granulocyte monocyte
colony-stimulating factor (GM-CSF) and mouse IL-7 were
purchased from R&D Systems (Minneapolis, MN, USA).
Flow cytometry and cell-sorting strategy
ONP cells were sorted from bone marrow of 2-week-old
C57BL/6J mice as previously described (Wang et al., 2006).
Analyses of cells were performed on a FACS Vantage SE
Turbo Sorter flow cytometer/sorter (Becton Dickinson, San
Jose, CA, USA) or a FacsCalibur cytometer (Becton
Dickinson), made available through the University Research
Core Facility. A lineage cocktail was prepared from a
mixture of APC-conjugated antibodies specific for CD-11b
(Mac-1), Gr-1, Ter-119, B220, CD19, CD3, CD4, CD8 and
sorted on FACS Vantage SE Turbo Sorter. Cultured cells
were stained using the appropriate mAbs and isotype-
matched antibodies were used as negative controls. All
samples were treated with an unlabeled anti-FcR (crystalliz-
able fragment receptor) to prevent inappropriate binding of
antibodies. Data analysis was accomplished using FlowJo
software (TreeStar, San Carlos, CA, USA).
A two-step culture strategy was used to grow the hematopoie-
tic progenitors. Sorted ONP cells were placed into 24-well
culture plates Costar, Cambridge, MA, USA containing
complete IMDM (Iscove’s Modified Dulbecco’s Media) sup-
plemented with 10% fetal bovine serum, 10 ng/ml recombi-
nant mouse SCF, 10 ng/ml recombinant mouse FL and 10 ng/
ml recombinant mouse IL-3 and cultured for the first 3 days.
After 3 days, cells were harvested and stained with B cell
surface marker (B220) and myeloid cell surface marker
(CD11b). These cultured cells were sorted into different wells
based on the positive or negative expression of these surface
markers. Cells were then cultured either with or without OP9
stromal cells. An additional 10 ng/ml exogenous IL-7 was
added to the culture media, and cells were cultured with OP9
stromal cells for another 9 days.
In a liquid medium culture system, ONP cells cultured for
3 days as mentioned above, without OP9 stromal cells
support, were washed and divided into two wells under two
different culture conditions. The first set of cultures con-
tained SCF, FL, IL-3 and IL-7 (10 ng/ml) to facilitate cell
differentiation to B lineage (thereafter referred to as culture
condition 1; Ito et al., 1996), and the second set of cultures
contained SCF, FL, IL-3 and GM-CSF (10 ng/ml) to facili-
tate cell differentiation along the myeloid pathway (referred
to as culture condition 2; Kondo et al., 2000). Cells were
then grown for another 9 days and fed every 2–3 days.
For single ONP cell culture, a single ONP cell was sorted
under 96-well culture plate (Costar) and cultured for 5 days
with the presence of SCF, FL and IL-3. After 5 days, cells
Alcohol and B-cell Development27
were splint into two wells and continuously cultured under
either culture condition 1 or 2 for another 12 days.
To determine whether alcohol affects the ONP cell at a
certain stage during cell differentiation, 100 mM alcohol was
added to the growth medium at different time points either
temporarily or permanently during the culture.
Quantitative real-time polymerase chain reaction analysis of
TFs (Pax5 and EBF) and the cytokine receptor (IL-7Rα)
RNA was prepared from ONP cells using an RNA extraction
kit (Qiagen Inc., Valencia, CA, USA). cDNA was generated
with the ThermoScript RT-PCR kit (GibcoBRL). Quantitative
real-time polymerase chain reaction (RT-PCR) was performed
using the Applied Biosystems Model 7700 sequence detection
system (Foster City, CA, USA). Primers and probes were
designed using Primer Express Software (Applied Biosystems)
and were as previously described (Wang et al., 2006). mRNA
for the TFs Pax5, EBF and cytokine receptor IL-7Rα was
quantitatively measured using Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) as an internal control. The relative
expression level of EBF, Pax5 and IL-7Rα versus GAPDH in
ONP cells was calculated by determining the ΔCtvalue for
each sample. The ΔCtvalue=(threshold cycle for the tran-
scriptional factor−the threshold cycle for GAPDH); i.e. the
number of additional cycles at which the sample reached the
threshold value after the GAPDH threshold value. Therefore,
the amount of input RNA for a given sample relative to
GAPDH is given by 2?DCt.
Student t-test and ANOVA of mean values were used in
comparison of differences in cell populations between the
alcohol and normal control groups and analyzed by a statisti-
cal software package (Instat, Graphpad Software, San Diego,
CA, USA). Differences between means were considered sig-
nificant when P <0.05.
Bipotential characteristic of ONP cells when cultured
ONP cells can be differentiated to either B cells or myeloid
cells with different culture conditions in vitro. As mentioned
in MATERIAL AND METHOD section, with two-step
culture conditions, after 12 days in culture, ONP cells differ-
entiated into B cells in liquid culture with the presence of
SCF, FL, IL-3 and IL-7. However, with the culture condition
more favored to myeloid cells, ONP cells generated into a
myeloid lineage (Fig. 1A and B).
To avoid any possible contamination, single ONP cells
were also sorted into 96-well culture plates and cultured for 5
days initially and then split into two wells. Cells in one well
were cultured under the condition 1 and the other well under
the condition 2. Cells were harvested after another 12 days in
culture and observed under the microscope (Fig. 1C and D).
Due to the ONP cell bipotential characteristics, it is impor-
tant to further investigate whether; (a) an intermediate stage
exists during the ONP cell differentiation that acts as a
branch point on B/myeloid cell divergence or (b) two differ-
ent cell populations developed during the ONP cell
differentiation and each cell population will eventually gener-
ate into a certain lineage. To answer this question, cells were
cultured under the stem cell reagents (SCF, FL and IL-3) for
3 days. After 3 days in culture, cells were stained with both
B cell surface marker B220 and myeloid cell surface marker
CD11b. Results showed after 3 days in culture, ONP cells
differentiated into two subpopulations. The majority of cul-
tured cells became B220−CD11b+, however, very few cul-
tured cells were still B220−CD11b−(Fig. 2A).
We then decided to sort these two cell populations separ-
ately and cultured each under the different liquid cell culture
conditions (either under culture condition 1 or condition 2).
Unfortunately, when sorted separately, only myeloid cells
were developed by B220−CD11b+cells. Both B220−CD11b+
and B220−CD11b−cells yielded no B cells no matter which
culture conditions were present (data not shown). However,
when B220−CD11b−and B220−CD11b+cells were mixed
together, and cultured under condition 1 in vitro, B cells
were generated after 12 days (Fig. 1A).
It is understood that in vitro B cell culture requires stromal
B220−CD11b+cells under the support of OP9 stromal cells
separately. After 9 days in culture, cells were harvested and
stained with B220 and CD11b. Our results showed only
B220−CD11b−cells generated B cells with the support of OP9
stromal cells (Fig. 2B). These above results indicated that ONP
cells first differentiated into two cell populations, which then
further grew into the different lineages. B220−CD11b−cells
can be differentiated into B cells, and B220−CD11b+cells can
be developed into myeloid lineage. During in vitro cell culture,
B220−CD11b+cells actually act as stromal cells to support
B220−CD11b−cells differentiation into a B lineage.
Alcohol affects the late stage of the ONP cells differentiation
into a B lineage
Previous laboratory results showed alcohol affects the ONP
cell differentiation into a B cell lineage both in vivo and
Fig. 1. ONP cells can differentiate into different lineages in vitro. ONP cells
can differentiate into both myeloid and lymphoid lineage. (A) showed ONP
cells yielded CD19+cells after 12 days bulk culture in liquid medium
containing SCF, FL, IL-3 and IL-7. However, when cultured in above
medium with SCF, FL, IL-3 and GM-CSF, ONP cells yielded only CD11b+
cells (B). (C) and (D) showed the different morphologies of clones derived
from a single ONP cell after cultured for 17 days with different culture
28Wang et al.
in vitro. Since ONP cells can further differentiated into either
B220−CD11b−cell that eventually generates into a B
lineage. It is important to know whether alcohol affects the
ONP cell in an early stage which occurs in the first 3 days or
affects the B220−CD11b−cell, which represents the late
stage of ONP cell differentiation to a B lineage. To address
this question, ONP cells were treated with 100 mM alcohol
for the first 3 days, then cultured cells were harvested and
stained with both B and myeloid cell surface marker (B220
and CD11b). Our results showed that there was no statisti-
cally significant difference between the alcohol-treated group
and normal control group on phenotype (Figs. 3A and 4A).
After 3 days of exposure to 100 mM alcohol, B220−CD11b−
cells were washed, stained and then sorted and cultured with
OP9 stromal cells in another 9 days without the addition of
alcohol, these cells yielded the same amount of B cells
(Figs. 3B and C and 4B and C). However, with continued
exposure to 100 mM alcohol during the whole course of in
vitro culture, B220−CD11b−cells yield very few B lineage
cells (Fig. 3D and E). This indicated that continued exposure
to alcohol is required to affect the ONP cells further differen-
tiation into a B lineage.
To demonstrate whether only exposure to alcohol at a late
stage affects B-cell differentiation; ONP cells were cultured
in vitro without the exposure of 100 mM alcohol for the first
3 days, then B220−CD11b−cells were sorted and cultured
under the condition 1 with the presence of alcohol. After
cells, and it is the
another 9 days in culture, cells were analyzed and the results
showed significant impairment of B-cell formation (Fig. 4D
and E), and there was no statistically significant difference
between the early alcohol-treated group receiving continued
alcohol (Fig. 3D and E) and late alcohol-treated group
(Fig. 4D and E).
Taken together, this indicated that alcohol will not affect
the early differentiation of ONP cells. Alcohol only affects
the differentiation of ONP cells that have already developed
into the B220−CD11b−stage.
B220−CD11b−cells were also further analyzed to confirm
these cells are not undifferentiated ONP cells. Our results
showed that these B220−CD11b−cells became c-Kit−cells
instead of the ONP cells with the positive expression of c-kit
(Figs. 3F and 4F). Negative expression of c-kit on cell
surface indicates the cell underwent further differentiation.
Alcohol down-regulated transcription factor and cytokine
receptor expression during ONP cell differentiation
One of the mechanisms by which progenitor B cells differ-
entiate into B lineage is the appropriate up-regulation of
several TFs and cytokine receptors. TF EBF, Pax5 and
cytokine receptor IL-7Rα play critical roles in development
of B cells. Failure to up-regulate EBF, Pax5 and IL-7Rα
results in impairment of B-cell differentiation. EBF is one
of the upstream TFs of Pax5, and inappropriate expression
of EBF results in the poor expression of Pax5. IL-7Rα
expression also can affect progenitor B cells’ expression of
EBF and Pax5. Therefore, it is important to find out
whether alcohol affects these TFs and/or cytokine receptor
In this study, the expression level of TF EBF, Pax5 and
cytokine receptor IL-7Rα were analyzed kinetically during
the in vitro liquid culture. According to the results mentioned
above, B220−CD11b+cells are not progenitor B cells but
acts as the supporting stromal cells, which are also essential
during B cell culture in vitro. Therefore, ONP cells were
sorted and cultured for 3 days. After 3 days, these cultured
cells were split into two groups and grew for another 9 days,
one group with the addition of 100 mM of alcohol under
culture condition 1, the other without the exposure of
alcohol under culture condition 1 as a normal control group.
The relative expression level of EBF, Pax5 and IL-7 were
measured on Day 0, 3, 6, 9 and 12 kinetically in the alcohol-
treated group and compared with the normal control group.
The results showed that alcohol affects the up-regulation of
TF EBF, Pax5 and IL-7Rα (Fig. 5) leading to the poor
differentiation of ONP cell into a B lineage. B220−CD11b+
cells were also cultured with or without the exposure of
alcohol, with the expression level of TF EBF, Pax5 and
IL-7Rα were also measured kinetically on Day 6, 9 and 12.
Results showed no change in expression levels of EBF, Pax5
and IL-7Rα between the alcohol and non-alcohol groups
(data not shown).
Although myeloid and lymphoid lineage cells have shown to
be derived from different hematopoietic precursors, some
previous studies have identified the existence of biphenotypic
Fig. 2. B220−CD11b−cells can differentiate into B cells with the support of
OP9 stromal cells. ONP cells were sorted and cultured with SCF, FL and
IL-3 for 3 days, phenotype analysis showed <5% cells differentiated into
B220−CD11b−cells (A). When sorted B220−CD11b−cells separately and
cultured with OP9 stromal cells for another 9 days, over 90% of these cells
differentiated into B cells (B).
Alcohol and B-cell Development29
myeloid/lymphoid cells (Ford et al., 1992; Graf et al., 1999;
Zeisig et al., 2003). The data present here demonstrate that
ONP cells can differentiate into both myeloid and lymphoid
lineages under different culture conditions. In this study,
cellswith multipotential propertieswere analyzed
phenotypically by anti-mouse B220 and CD11b after culture
in a timely and ordered manner. The observations obtained
from our experiments illustrated that these cells first differen-
tiated into two cell populations; B220−CD11b−precursors,
which differentiatedintoa lymphoidlineage and
Fig. 3. Alcohol affects the late stage of the ONP cell differentiation into a B lineage. ONP cells were sorted and first cultured with SCF, FL and IL-3 for 3
days. After 3 days in vitro culture, cells were differentiated into two populations: B220−CD11b−and B220−CD11b+cells (A). These B220−CD11b−cells
started to lose their surface expression of c-Kit (F). B220−CD11b−cells were sorted and ‘splint’ into two wells, cells in one well were cultured with OP9
stromal cells without alcohol for another 9 days and yielded B220+cells (B). These B220+cells were further stained with anti-CD19 (B-cell marker) showed
over 90% of CD19 positive expression (C). Cells in the other well were cultured with OP9 stromal cells with the addition of 100 mM of alcohol for another 9
days. Very few cells differentiated into a B lineage (D) with minimal expression of CD19 (E).
Fig. 4. Alcohol does not affect the early stage of the ONP cell differentiation. ONP cells were sorted and cultured with SCF, FL, IL-3 and 100 mM of alcohol for
the first 3 days. Cells were analyzed after 3 days and showed two cell populations: B220−CD11b−and B220−CD11b+cells (A). There is no statistically significant
difference between early alcohol-treated and normal control groups (P>0.05, A and Fig. 3A). These B220−CD11b−cells also lost their surface expression of
c-Kit (F). After 3 days of culture, B220−CD11b−cells were also sorted and ‘split’ into two wells, cells were all co-cultured with the support of OP9 stromal cells.
Cells in one well without the exposure of alcohol were differentiated into B220+cells (B) with strong CD19 expression (C) and reached no statistically significant
difference (B, C versus Fig. 3B and 3C P>0.05). Cells in the other well with the addition of 100 mM of alcohol yielded no B cells (D and E).
30 Wang et al.
B220−CD11b+precursors, which differentiate into myeloid
Moreover, in vitro culturing of progenitor B cells requires
stromal cell support (Rolink et al., 2000). Cells without
stromal cell support will either undergo apoptosis or failure
to further differentiate into a B lineage (Crooks et al., 2000;
Gimble et al., 1993; Nutt et al., 1999; Rolink et al., 2000).
Our results showed that ONP cells first differentiated into a
B220−CD11b−cells. It is the B220−CD11b+cells, which
function as stromal cells, supporting the B220−CD11b−cells
differentiation into B cells.
Previous studies showed that alcohol can affect the ONP
cell differentiation into a B lineage both in vitro and in vivo
(Wang et al., 2006, 2009). However, it is important to know;
(a) whether alcohol affects cell differentiation at an early
stage, late stage or on both stages, (b) does the effect of
alcohol on B-cell differentiation temporary or permanent and
(c) the mechanism by which alcohol affects the progenitor
To determine whether alcohol affects cell differentiation at
an early or late stage, 100 mM of alcohol was added at
different time points during the cell culture. The100 mM
concentration of alcohol was used in this study, because it
has no direct cytotoxicity to ONP cells and will not affect
the proliferation of ONP cell cultures in vitro (Wang et al.,
2006, 2009). Results of this experiment showed that alcohol
does not affect the early differentiation of ONP cells until the
B220−CD11b−stage. Alcohol-exposed B220−CD11b−cells
were not the same as undifferentiated ONP cells since they
started to lose the expression of SCF receptor (c-kit) on their
surface indicating cells underwent further differentiation.
However, our results showed that the exposure of 100 mM
alcohol affects the ONP cell differentiation to a B lineage.
Significant reduction in B lymphocytes after 12 days indi-
cates that alcohol affects the ONP cell differentiation in the
late stage only. The effect of alcohol on the late stage ONP
cell differentiation was permanent.
Further investigation on the molecular basis of B lineage
commitment was performed by measuring the transcription
factor EBF and Pax5 and cytokine receptor IL-7Rα among
these ONP cells. EBF and Pax5 are believed to be the impor-
tant TFs in the development of B lymphocytes (Busslinger
et al., 2000; Medina and Singh, 2005). Previous studies
showed that maturation of B cells was severely blocked in B
progenitors of transgenic mice lacking of EBF or Pax5 (Kee
and Murre, 1998; Nutt et al., 1999). It is still uncertain
whether cells will strictly differentiate into B lineage with the
Pax5 gene turned on. Current experimental evidence indi-
cates that Pax5 expression within cells does not block the
early myeloid lineage, or natural killer cell development
(Cotta et al., 2003). Moreover, Pax5 can be reversibly
switched in immature hematopoietic progenitors (Okubo
et al., 2002). Based on the results of these studies, it is poss-
ible that the expression of TFs can be dynamically modu-
lated within multipotential progenitors by environmental
factors in favor of a specific lineage commitment. Our find-
ings seem to support this hypothesis. Among ONP cells,
directly sorted from mouse bone marrow, EBF and Pax5
message RNA were measured by using quantitative real-time
RT-PCR. After 3 days, culture with the addition of stem cell
reagent including SCF, FL and IL-3, which favors the
myeloid lineage in vitro, both EBF and Pax5 are down-
regulated. Compared with their phenotypes, the majority of
cultured cells showed CD11b positive as well. With exposure
to IL-7, which favors B lineage commitment, EBF and Pax5
were up-regulated rapidly. Meanwhile, without the addition
of IL-7, Pax5 was undetectable. This dynamic switch of the
expression level of EBF and Pax5 is likely to address, at
least partially, the molecular basis of B lineage differentiation
in ONP cells. However, the addition of 100 mM of alcohol
to an in vitro culture down-regulates the expression level of
TF EBF, causing minimal expression of Pax5. Low Pax5
expression results in the paucity of B-cell commitment.
In conclusion, our data shows that development of
myeloid or lymphoid cells from ONP cells diverges through
B220−CD11b+/ B220−CD11b−branch points. Expression of
transcription factor EBF and Pax5 does not block early
myeloid lineage commitment. The microenvironment that
favors the specific lineage differentiation seems to play a
critical role. With the exposure of IL-7, EBF and Pax5 rever-
sibly switch to a high level and progenitor cells generate a B
lineage. However, the exposure to 100 mM of alcohol, at a
late stage of ONP cell differentiation in vitro, severely blocks
the expression of TFs, resulting in impairment of B-cell
differentiation. Taken together, these results may indicate the
common developmental pathway and the molecular basis of
myeloid/lymphoid divergence among ONP cells.
Fig. 5. Alcohol down-regulated the transcription factor expression during the ONP cell differentiation. ONP cells were harvested at day 0, 3, 6, 9 and 12
during in vitro culture in both normal control and alcohol groups. The expression level of EBF, Pax-5 and IL-7Rα were analyzed using real-time PCR. Results
showed the relative expression levels of TFs EBF (A), Pax5 (B) and cytokine receptor IL-7Rα (C) were increased kinetically in the control group but not in
the alcohol group.
Alcohol and B-cell Development31
Funding — This work was supported in part by grants from the National Institutes of Download full-text
Health R01 AA-14141 and Louisiana State University Health Science Center
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