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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2823-2829
A Regulatory Role for Fc Receptors CD16 and CD32 in the
Development of Murine B Cells
By
Belen de Andres,
Allan L. Mueller,
Sjef Verbeek,
Matyas Sandor, and
Richard G. Lynch
From the Department of Pathology, University of Iowa, Iowa City, IA;
the Department of Immunology, University Hospital Utrecht, Utrecht, The
Netherlands; and the Department of Pathology and Laboratory Medicine,
University of Wisconsin, Madison, WI.
 |
ABSTRACT |
Early in development, murine B-lineage progenitor cells express two
classes of IgG Fc receptors (Fc R) designated as FcRII (CD32) and
FcRIII (CD16), but mature B lymphocytes only express FcRII
(CD32), which functions as an inhibitor of B-cell activation when it is
induced to associate with mIgM. The functions of CD16 and CD32 on
B-lineage precursor cells have not previously been investigated. To
search for FcR functions on developing B-lineage cells, normal
murine bone marrow cells were cultured in the presence of 2.4G2, a rat
monoclonal antibody that binds to CD16 and CD32, or in the presence of
control normal rat IgG, and then the B-lineage compartment was analyzed
for effects. Cultures that contained 2.4G2 showed enhanced growth and
differentiation of B-lineage cells compared with control cultures. The
enhancing effect of 2.4G2 also occurred when fluorescence-activated
cell-sorted B-cell precursors (B220+,
sIgM , HSAhigh,
FcR+) from normal bone marrow were cocultured with
BMS2, a bone marrow stromal cell line, but not when they were cultured
in BMS2-conditioned media. The enhancement of B-lineage development
induced by 2.4G2 was CD16-dependent and CD32-dependent, because 2.4G2
did not effect B-lineage growth or differentiation in cultures of bone
marrow from mice in which either the gene encoding CD16 or CD32 had
been disrupted. Analysis of fresh bone marrow from the CD16
gene-disrupted mice showed normal numbers and distribution of cells
within the B-cell compartment, but in CD32 gene-disrupted mice, the
B-cell compartment was significantly enlarged. These experiments
provide several lines of evidence that the FcR expressed on murine
B-cell precursors can influence their growth and differentiation.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
Fc RECEPTORS DESIGNATED as FcR bind to
epitopes located in the constant region domains of IgG and are
expressed on cells of hematopoietic lineages in which they mediate
multiple physiological functions.1,2 Most previous studies
of FcR function have focused on mature cells and have identified a
diversity of roles for FcR on macrophages, polymorphonuclear
leukocytes, natural killer (NK) cells, and lymphocytes.
Although FcR are expressed very early in hematopoietic cell
ontogeny,3-7 the functional significance of these receptors
on hematopoietic progenitor cells is unknown.
During murine lymphopoiesis, T- and B-lineage progenitor cells express
FcRIII (CD16) and FcRII (CD32), but with the initiation of
antigen receptor gene rearrangement, CD16 is downregulated in B cells
and T cells, and CD32 is downregulated in T cells.8 These
curious patterns of FcR displayed on developing lymphoid cells raise
the possibility that CD16 and CD32 may participate in some aspect of T-
and B-cell development before the stage at which these lymphoid cells
become antigen responsive.
That multiple functions have been associated with FcR on mature
myeloid and lymphoid cells can be partially accounted for by the
structural heterogeneity of FcR molecules. The chemical structures
of members of the family of FcR show interesting similarities and
striking differences, characteristics that have important functional
implications. There is 95% identity in the amino acid sequences of the
ligand-binding, extracellular segments of FcRII and FcRIII, the
two low-affinity IgG receptors expressed on lymphocytes, but the
sequences of the transmembrane and cytoplasmic signaling segments are
structurally unrelated.9,10 The cytoplasmic segment of CD16
associates with a homodimer of an activation signaling molecule termed
FcR, which is a member of the CD3 chain family.11 The
-chain contains an ITAM (immunoreceptor tyrosine activating motif)12 that, when tyrosine phosphorylated, transduces
cellular activation signals. The cytoplasmic segment of CD32 contains a phosphorylable tyrosine in a 13 amino acid motif designated an ITIM
(immunoreceptor tyrosine inhibitory motif) that inducibly associates
with the tyrosine phosphatase SHP-1 and can inhibit ITAM-induced
cellular activation.13,14
We were interested in the functional significance of FcR on
progenitor B-lineage cells because, in a previous study,5 FcR on murine fetal prothymocytes had been shown to influence the
rate of differentiation to fully mature, immunocompetent thymocytes. In
those experiments, the addition of an anti-FcR monoclonal antibody
(MoAb; 2.4G2) to fetal thymic organ cultures resulted in a
dose-dependent acceleration in thymocyte maturation to   TCR+, HSAdim, CD3bright T cells.
2.4G2 recognizes an epitope in the extracellular segment of the FcR
and does not distinguish between CD16 and CD32. Acceleration of
thymocyte maturation was also induced by the addition of soluble recombinant FcR to the fetal thymic organ cultures. This combination of findings raised the possibility that FcR on pro-T cells normally interacted with alternative non-Ig ligand(s) and that this interaction had the effect of modulating the rate of differentiation of
prothymocytes to mature T cells. Experimental blockade of this putative
interaction, by the addition of anti-FcR MoAb or soluble recombinant
FcR to the thymic organ cultures, resulted in a faster rate of
T-cell maturation. In support of the alternative ligand hypothesis was our finding that soluble recombinant FcR bound strongly to a small
population of CD44, CD3 thymic
stromal cells.5 Additional evidence for a role of CD16 in
modulating the maturation of prothymocytes was the finding that
overexpression of CD16 on prothymocytes in mice expressing an
FcR-chain transgene driven by a CD2 promoter was associated with a
decreased rate of differentiation to mature thymocytes.15
Because FcR are also expressed on B-lineage precursor
cells16 and because we had observed that soluble
recombinant FcR could bind to normal murine bone marrow stromal
cells and to BMS2, a murine bone marrow stromal cell line,5
we considered the possibility that FcR on B-cell precursors might
influence the development of B-lineage cells. In the studies reported
below, we found that the addition of 2.4G2 accelerated B-lineage
lymphopoiesis in cultures of bone marrow from normal mice, but not in
cultures of bone marrow from CD16 or CD32 knockout mice. These studies provide experimental evidence that FcR on developing B-lineage cells
can modulate B-cell development.
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MATERIALS AND METHODS |
Cultures of bone marrow from normal mice.
C57Bl6/129-CD16 gene-disrupted mice (CD16/)
were constructed by Dr Sjef Verbeek (Department of Immunology,
University of Utrecht, Utrecht, The Netherlands).17
C57Bl6/129-CD32 gene-disrupted mice were purchased from
Taconic (Germantown, NY). Caged mice were maintained in a
horizontal laminar flow cabinet and provided with sterile food and
water. Femoral and tibial bone marrow samples were taken from 6- to
9-week-old mice, and a single-cell suspension was prepared in Hanks'
balanced salt solution (HBSS) following a previously described
procedure,18 with modifications. After three washes, 2 × 105 cells/mL were resuspended in RPMI 1640 supplemented with 10% bovine calf serum, 0.2 mmol/L L-glutamine, 0.1 mmol/L essential and nonessential amino acids, 0.1 mmol/L Na-pyruvate,
5 × 105 mol/L 2-ME, and an
antibiotic-antimycotic. All cell culture reagents used in this study,
unless otherwise specified, were purchased from GIBCO/BRL (Grand
Island, NY). In some experiments, bone marrow cells in 24-well plates
(#3424; Costar, Cambridge, MA) were cultured in the presence of 10 U/mL
recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF),
20 U/mL recombinant interleukin-5 (rIL-5), and 10 U/mL
rIL-3 (Genzyme, Cambridge, MA).18 In other experiments, a
media formulation containing IL-7 was used to promote the growth of B
cells.19 Viability of the cultured cells was determined by
Trypan blue dye exclusion.
MoAbs.
The MoAbs used in the fluorescence-activated cell sorting
(FACS) analyses were labeled with fluorescein
isothiocyanate (FITC), phycoerythrin (PE), or cyanine 5.18 (Cy5). They
included RB6-8C5 (a rat IgG2b reactive with the granulocyte marker,
Gr-1), 6B2 (a rat IgG2a antimurine CD45R/B220), Mac-1 (a rat IgG2b
antimurine CD11b), c-kit (a rat IgG2b antimurine CD117), S7 (a rat IgG
antimurine CD43), and M1/69 (rat IgG2b antimurine CD24, HSA; all
purchased from Pharmingen, San Diego, CA); and antimouse IgM (a hamster IgG; purchased from Jackson ImmunoResearch, West Grove, PA). In bone
marrow culture experiments, 2.4G2, a rat IgG2b antimurine FcRII/RIII,20 was added to the cultures (25 µg/mL). In
some experiments, a control rat IgG (purified rat IgG2b; Pharmingen) was added; in other experiments, the control was cytokines alone. Both
antibodies had previously been dialyzed against phosphate-buffered saline (PBS) and filtered under sterile conditions before
addition to the cultures. Endotoxin was not detected in 2.4G2 MoAbs by the Lumulus amebocyte lysate assay (Pyrochrome, Cape Cod, MA).
In some experiments, we used these MoAbs to determine the size of the
B-lineage subpopulations as defined by Wasserman et al.27
Flow cytometric analysis.
Cells were suspended at a concentration of 107 cells/mL in
HBSS buffer containing 10% bovine calf serum, 10 mmol/L HEPES, and 0.02% Na-azide (FACS staining buffer). Cells were stained in 50 µL
of conjugated Abs for 40 minutes at 4°C, washed three times, fixed
in 2% paraformaldehyde in PBS (pH 7.3), and analyzed on a Becton
Dickinson FACS Vantage (Becton Dickinson, Mountain View, CA) equipped
with four decade logarithmic amplifiers. Forward and side scatter and
two or three fluorescence parameters were collected on 10,000 cells and
the data were analyzed on a VAX 4000 computer equipped with DESK
software.16 For sorting pro/pre-B cells
(B220+, IgM), 2 × 107
cells/mL were stained with anti-B220 and anti-IgM in FACS staining buffer. Cells were collected on a Coulter Epics 753 flow cytometer (Coulter, Hialeah, FL) and analyzed using Coulter Elite
software (University of Iowa College of Medicine Flow Cytometry
Facility, Iowa City, IA). Reanalysis of the sorted populations showed a purity greater than 98%.
Cocultures of progenitor B cells with stromal cell lines.
Sorted progenitor B cells (B220+, IgM,
HSAhi, 2 × 105 in 2.0 mL) were cultured
for 3 days with the stromal cell line BMS2 generously provided by Dr
P.W. Kincade (Oklahoma Medical Research Foundation, Oklahoma City, OK).
The BMS2 stromal cell line was maintained in complete media and plated
into 6-well plates (Costar 3406) at a density of 1 × 105 cells per 6-cm2 well and expanded to 80%
confluence to support pre-B-lymphocyte growth.21,22
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RESULTS |
B-lymphopoiesis and eosinophilopoiesis are induced in bone marrow cells
cultured with IL-3, IL-5, and GM-CSF.
After 72 hours of culture in the presence of GM-CSF, IL-3, and IL-5,
the B220+ population had increased approximately 1.5-fold,
and about 43% of the cultured cells expressed B220
(Table 1). The number of B220+/IgM+ cells had increased approximately
threefold, and these accounted for about one third of the cultured
cells (Table 1). Microscopic examination of Wright-Giemsa-stained
smears showed that bone marrow cells freshly isolated from normal, 6- to 9-week-old mice contain less than 5% eosinophils, but in the
presence of GM-CSF, IL-3, and IL-5 this had increased to approximately
20% by 72 hours of culture. This cytokine protocol was being used
because we were investigating the role of FcR in eosinophilopoiesis
when the initial observation of the effect of 2.4G2 on B-cell
development was made. Under the conditions of culture used, virtually
all the Gr-1+ cells present at 72 hours were
eosinophils.23 Neutrophils were not detected by
Wright-Giemsa microscopic examination of smears. In addition,
uncommitted progenitors and stromal cells (c-kit+ cells)
account for approximately 46% of the cultured cells at 72 hours. The
percentage of cells expressing T-cell markers at 72 hours was 5% or
less.
2.4G2 accelerates the differentiation of progenitor B cells in bone
marrow cultures.
To determine if B-lymphopoiesis is influenced by FcR interactions,
bone marrow cells were cultured in the presence of IL-3, IL-5, and
GM-CSF and 25 µg/mL of the anti-FcR MoAb (2.4G2) to engage the
FcRs CD16 and CD32. As shown in Table 1, cultures that contained
2.4G2 had approximately 50% more B220+ cells and
B220+/IgM+ cells compared with cultures not
containing 2.4G2. To further examine the effect of 2.4G2 on
B-lymphopoiesis, additional markers were assessed by FACS analysis.
Figure 1 shows a representative flow
cytometric analysis of bone marrow cells after 3 days of culture with
cytokines in the presence or absence of 2.4G2. In addition to the
significant increment in the size of the B220+/IgM+
population induced by treatment with 2.4G2, Fig 1 shows that bone
marrow cells cultured with cytokines and 2.4G2 showed an enhanced
expression of CD19 and HSA and that the level of B220 on individual
cells was increased. In data not shown, the
B220+/mIgM+ cells did not express mIgD, CD23,
or CD25. Taken together, these findings indicate that 2.4G2 promoted
differentiation of B-cell precursors to the stage of the immature B
cell.24,25
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| Fig 1.
Effect of 2.4G2 on B220+ cells in bone
marrow cultures treated with IL-3, IL-5, and GM-CSF. Bone marrow cells
(2 × 105 cells per well) were cultured with cytokines for
3 days in the presence of 2.4G2 (25 µg/mL) or with cytokines alone.
The analysis shown was performed after setting a lymphocyte gate based
on orthogonal and forward scatter. The figure shows one representative
experiment of three with similar results.
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Effect of 2.4G2 on the growth and differentiation of bone marrow
B-lineage cells containing disrupted CD16 or CD32 genes.
Normal pro/pre-B cells are known to express FcRII1(CD32) and
FcRIII(CD16), but FcRIII is detected only during the
developmental stages before the expression of mIgM (Hagen et al,
submitted for publication). In contrast, FcRII is
present throughout B-cell ontogeny, even to the plasma cell
stage.16 To determine whether the 2.4G2-triggered
acceleration of B-lineage development in vitro depended on the
expression of CD16 and/or CD32, experiments were conducted with
bone marrow cells obtained from mice in which the CD16 gene or the CD32
gene had been disrupted. The CD16/ mice do
not produce the -chain subunit of FcRIII, but they do produce the
-chain subunit that is also a subunit of other immunoreceptors.17 The CD32/ mice
do not produce CD32 chains.26 Following the same
protocol as that described above, bone marrow cells from
CD16/ or CD32/ mice
were cultured with and without 2.4G2. We found that, in the bone marrow
cultures from these mice, there was no significant effect of 2.4G2 on
B-lineage growth or differentiation (Table 1). By 72 hours of culture,
there was a decrease in the total number of B220+ cells,
but not of B220+/IgM+ cells, in the samples
from CD16/ and
CD32/ mice. Thus, the loss of B-lineage cells
in the cultures from CD16/ and
CD32/ mice appears to have been selective for
cells at stages before the expression of mIgM. The absence of an effect
of 2.4G2 in the bone marrow cultures from
CD16/ and CD32/
mice suggests that FcR may play a previously unsuspected role in
B-lymphopoiesis.
It was possible that the absence of any effect of 2.4G2 on the
B-lineage cells from CD16/ and
CD32/ mice and the decrease in
B220+ cells in the cultures of bone marrow from these mice
were reflections of some defect in the culture system. We thought that
this possibility was unlikely because, in the cultures of the
CD16/ marrow, we found the same high level of
eosinophil production as in the bone marrow cultures from normal mice.
This indicted that myelopoiesis was proceeding normally in those
cultures. However, because IL-7-containing media is the more
conventional method to promote B-lymphopoiesis in vitro, we conducted
additional experiments incorporating IL-7 into the cultures. The
presence of IL-7 promoted a more robust level of B-lymphopoiesis
(Table 2). Under these conditions of
culture, the presence of 2.4G2 resulted in further increases in
B220+ cells and B220+/IgM+ cells.
To further explore this consideration, we examined B-lineage cells from
normal, CD16/, and
CD32/ mice for their expression of FcR as
detected by FACS analysis of 2.4G2-binding cells. As shown in
Fig 2, almost 90% of the B220+
cells in the bone marrow from normal background strain mice bind 2.4G2,
but only 40% of the B220+ cells from
CD32/ bind 2.4G2. These data suggest that
normally CD16 is coexpressed with CD32 on some B-lineage cells,
because, in its absence in CD16/ mice, 2.4G2
continues to detect approximately the same percentage of
B220+ cells. However, the data also suggest that CD32 is
expressed on a large fraction of B220+ cells that do not
coexpress CD16, because, in the CD32/ mice,
there is a large fraction of B220+ cells that are not
detected with 2.4G2. In the CD32/ mice, the
vast majority of the B220+ cells that bind 2.4G2 belong to
the low-density B220+ population, indicating that CD16 is
expressed on the less mature B-lineage cells. This finding agrees well
with data on CD16 and CD32 transcript expression in a large panel of
B-lineage tumor cell lines and FACS-sorted normal B-lineage
subpopulations (Hagen et al, submitted for publication).
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| Fig 2.
FcR expression on B220+ cells. Flow
cytometric analysis of fresh bone marrow cells from
wild-type, CD16/, and
CD32/ mice. The lower part of the figure shows the
percentage of B220+ cells binding 2.4G2.
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2.4G2 enhances the differentiation of progenitor B lymphocytes
cocultured with stromal cells.
Because CD16 and CD32 can be expressed on many different cell types in
bone marrow, the experiments and data presented above, although showing
a requirement for CD16 and CD32 in the enhancement induced by 2.4G2,
did not address on which cells the CD16 and CD32 expression was
required. Because the previous studies with fetal
thymocytes5 suggested that it was the FcR on the
prothymocyte that was required for the 2.4G2-triggered promotion of
thymocyte maturation, we investigated whether 2.4G2 would enhance the
maturation of purified pro-B cells (B220+,
IgM, FcR+) cultured with a bone
marrow stromal cell line that supports B-lineage development in
vitro.21 Experiments were performed using FACS-sorted
normal B-cell precursors (B220+, IgM,
FcR+, CD43+, HSAhi) cultured
with BMS2, a murine bone marrow stromal cell line. As shown in
Fig 3, during 3 days of cocultures with
BMS2 cells, the pro-B cells underwent further differentiation, as
reflected by the shift from HSAhi to HSAlo and
the shift from IgM to IgM+. However, in
the cultures that contained 2.4G2, there was a statistically significant enhancement of IgM expression
(Table 3). This was manifested by both an
increase in the percentage of IgM+ cells and a higher level
of IgM per cell (Table 3). When the purified pro-B cells were cultured
alone in normal media or in BMS2-conditioned media, the pro-B cells
were nonviable after overnight culture (data not shown).
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| Fig 3.
The effect of 2.4G2 on differentiation of B-lineage
precursor cells (B220+, IgM,
HSAhi). Fresh FACS-sorted B220+,
IgM bone marrow cells (left panel) were cocultured with
bone marrow stromal cell line BMS2. IgM and HSA expression on
B220+ cells after 72 hours of culture without (middle
panel) or with 2.4G2 (25 µg/mL; right panel). Cultures with 2.4G2
showed an enhanced percentage of cells with IgM expression and
increased amounts of IgM per cell.
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Table 3.
Effect of 2.4G2 on Purified Progenitor B Cells
(B220+, IgM) Cocultured With Bone Marrow
Stromal Cells (BMS2) for 72 Hours
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As was found in the experiments with cultures of unfractionated bone
marrow cells, the enhancing effect of 2.4G2 on the purified pro-B cells
cocultured with BMS2 cells was CD16-dependent. The data in Table 3 show
that the B-precursors purified from CD16/
bone marrow were not influenced by the presence of 2.4G2 when cocultured with BMS2 stromal cells. Although the B-cell precursors from
CD16/ bone marrow did differentiate to
HSAlo, IgM+ cells when cocultured with BMS2
cells, differentiation was not affected by the presence of 2.4G2. In
comparable experiments performed with purified pro-B cells from
CD32/ bone marrow, the presence of 2.4G2 did
not influence B-cell differentiation (data not shown).
Because the data from the in vitro experiments with normal bone marrow
(Fig 1 and Table 1) suggested that FcR could influence B-cell
development, it was of interest to consider the in vivo situation. To
begin to address this issue, we measured the expression of B220, CD19,
and HSA and determined the sizes of the Hardy fractions A, B, C, D, E,
and F27 in fresh bone marrow samples from
CD16/, CD32/ and
normal control mice of the C57Bl6/129 background strain. The results of
these experiments are shown in Table 4. No
differences were seen between the CD16/ and
normal background control samples, but in the
CD32/ samples there was a significant
increase in total numbers of B-lineage cells (CD19+) and in
the size of the Hardy fractions D through F.
| |
DISCUSSION |
The major finding of the present study is that CD16 and CD32, the
low-affinity FcR expressed on normal murine pro/pre-B cells as well
as on many other hematopoietic cells, can influence the growth and
differentiation of developing B-lineage cells. These findings identify
a possible role for FcR in B-cell ontogeny, and they parallel the
original findings of Sandor et al5 that FcR on
prothymocytes can influence T-cell ontogeny. In these studies, the
presence of anti-FcR antibody (2.4G2), but not control rat IgG
antibody, in cultures of normal bone marrow cells enhanced the growth
of B-lineage precursor cells
(B220+/HSAhi/mIgM) and
promoted their differentiation to more mature levels
(B220+/HSAlo/mIgM+). Because these
mIgM+ cells did not express mIgD, CD23, or CD25 (data not
shown), their phenotype is characteristic of the stage that has been
defined as the immature B cell.24,25 Although modest in
magnitude, the enhancing effect of 2.4G2 in vitro is reproducible and,
as shown above, occurs in several different experimental formats. Furthermore, the expansion of the B-lineage compartment in the bone
marrow of CD32/ mice suggests that our in
vitro findings have an in vivo relevance.
There are several possible mechanisms that could account for these
findings. One possibility is that 2.4G2 binds to FcR on B-lineage
precursor cells and directly stimulates the cells to proliferate and
differentiate. Because 2.4G2 binds to both FcRII (CD32) and
FcRIII(CD16), the enhancing effect of 2.4G2 could be dependent on
CD32, CD16, or both. The data from experiments with bone marrow from
CD16/ and CD32/
mice indicate a requirement for both CD16 and CD32. Because CD16 and
CD32 are coexpressed only during the early stages of B-lymphopoiesis, it is possible that 2.4G2 triggers an interaction between CD16 and CD32
on B-cell progenitors that results in enhanced B-lymphopoiesis. Alternatively, the 2.4G2-induced enhancement might be mediated by a
sequential mechanism in which an initial signal involving both CD16 and
CD32 on the progenitors was followed by a second signal through the
solitary CD32 on the more differentiated cells.
Another possibility is that the enhancement effect is mediated
indirectly by FcR+ cells that are not B-lineage
precursors. Because CD32 and CD16 are expressed on other types of
hematopoietic cells,28 it was possible that, when 2.4G2
bound to CD32 and/or CD16 on non-B-lineage cells in the
cultures, those cells promoted the growth and differentiation of the
B-lineage cells. Although not formally eliminated as a possible
mechanism, an indirect effect seems unlikely, because enhancement
occurred in the experiments in which FACS-sorted B-cell precursors were
cocultured with cells of BMS2, a murine bone marrow stromal cell line.
In these experiments, a requirement for the presence of both CD16 and
CD32 was demonstrated by using FACS-sorted B-cell precursors from
CD16/ and CD32/
mice.
A third possible mechanism to account for the enhanced differentiation
induced with 2.4G2 is that the anti-FcR antibody blocks interaction
between FcR on the B-cell precursors and an alternative, non-Ig
ligand on bone marrow stromal cells. In the experimental system we
used, the cultured bone marrow cells are supported by components of the
media, including the added GM-CSF, IL-3, and IL-5,18 and by
soluble factors produced by cells in the culture, particularly by the
hematopoietic stromal cells.21 Several observations suggest
that direct contact of the precursor B cells and stromal cells is
required for the enhancing effect of 2.4G2. In previous studies,5 it was shown that soluble, recombinant FcR
binds to the surface of BMS2 cells, a finding that implies the presence of an alternative, non-Ig counter-receptor for FcR on BMS2 cells. It
has also been shown that normal murine bone marrow stromal cells bind
soluble, recombinant FcR.8 The presence of a receptor for FcR would provide a receptor:counter-receptor couple that could
mediate direct interaction between B-lineage precursors and stromal
cells. A requirement for direct contact between the B-precursors and
stromal cells is also favored by the finding that, although the murine
bone marrow stromal cell line BMS2 supports the growth of B-lineage
precursors, BMS2-conditioned media does not. In data not shown, we
found that the conditioned medium generated by culturing BMS2 cells for
24 hours in cytokines plus 2.4G2 was unable to support the survival of
FACS-sorted normal B-precursors. Nonetheless, the mechanism that
mediates 2.4G2 triggered enhancement of B-lymphopoiesis in this model
is unknown and awaits further investigation. There are interesting
parallels between developing T and B cells with regards to the
expression of FcR. In both lineages the onset of rearrangement of
genes that encode antigen receptors coincides with the downregulation
of CD1629-31 (Hagen et al, manuscript
submitted). Thus, with the exception of some classes of
 T cells, CD16 is not expressed on mature T cells32 or mature B cells. This strongly implies that CD16 mediates its function(s) at the early stages of T- and B-lymphopoiesis. The other
interesting parallel is that the presence of anti-FcR antibody in
fetal thymus organ cultures5 and cultures of pro/pre-B
cells accelerates growth and differentiation of T- and B-lineage cells, respectively. This is particularly interesting in view of the report33 that human macrophage differentiation is also
accelerated in vitro by antibodies directed to FcRI (CD64), the
high-affinity IgG receptor expressed on monocyte precursors. Based on
previous studies, we proposed that experimental blockade of the
interaction between FcR on prothymocytes and a putative alternative
ligand on stromal cells accelerates maturation of the prothymocyte. If correct, this model predicts that overexpression of FcR would retard
prothymocyte development. This prediction has been confirmed for
T-lineage development by Flamand et al15 in
mice constructed with a CD16 -chain transgene driven by a CD2
promoter.
Curiously, the patterns of expression of CD16 and CD32 on progenitor
and immature T and B cells show a striking concordance, whereas their
display on adult T and B cells is discordant. This is interesting,
because the ligand-binding properties of CD16 and CD32, although
distinct, are predicted to have considerable overlap based on the 95%
identity in amino acid sequences of their extracellular, ligand-binding
segments.34 Because CD16 and CD32 are structurally
unrelated in their transmembrane and cytoplasmic segments, different
cellular consequences follow the binding of ligand by CD16 and CD32.
The cytoplasmic segment of the ligand-binding -chain of CD16
associates with a homodimer of the FcR -chain, an ITAM-containing
signal transducing molecule.12,35,36 It is known from the
study of CD16 on other cells that the binding of ligand by CD16
triggers a cascade of activation events mediated through specific
tyrosine kinases. Disappearance of the ITAM-containing CD16 from the
developing T and B cells coincides with the acquisition of
antigen-responsiveness that is mediated through another set of
ITAM-containing molecules, the TCR and BCR, respectively. Although CD16
is not expressed on adult T and B cells, ligands for CD16 still are
present in the host and CD16 continues to be expressed on other cells,
such as NK cells. Thus, the developmental window during which FcRIII
(CD16) is expressed on B-precursors allows it to influence their
development only before antigen-specific clonal commitment. In
contrast, CD32 is present on progenitor and mature B-lineage cells, but
it does not associate with the FcR -chain. When induced to associate
with mIgM on mature B-lymphocytes, CD32 blocks B-cell
activation.37 The molecular basis of the blockade of B-cell
activation has been shown to be an ITIM,12 located in the
cytoplasmic segment of CD32 that functions to inhibit phosphorylations
triggered by the ITAM-containing immunoreceptors TCR, BCR, and
FcR.13 Whether CD32 mediates an inhibitory function in
progenitor T- and B-lineage cells has not been investigated. In view of
the inhibitory effects mediated by CD32 in mature cells of several
hematopoietic lineages,13 it is interesting that fas-dependent programmed cell death is induced in developing and mature
murine eosinophils by aggregation of CD32, but not CD16, on their cell
surface.38
Finally, the occurrence of FcR on T- and B-cell precursors and their
ability to influence lymphopoiesis has implications for certain
diseases. For example, patients with multiple myeloma39 and
human immunodeficiency virus (HIV)40 develop high
circulating levels of soluble FcR. Conceivably, these soluble
receptors could competitively inhibit the generation of signals through
FcR on developing lymphoid cells, thereby uncoupling lymphopoiesis.
In addition, circulating IgG immune complexes in certain immunological disorders might avidly bind to FcR on developing lymphoid cells, thereby triggering pathologic signaling and altered immune function.
| |
FOOTNOTES |
Submitted January 7, 1998;
accepted June 13, 1998.
Address reprint requests to Richard G. Lynch, MD, Department of
Pathology, College of Medicine, 1117 Medical Laboratories, University
of Iowa, Iowa City, IA 52242; e-mail: richard-lynch{at}uiowa.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge the excellent secretarial assistance
of Vicki Brown.
| |
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Abstract
Introduction
Abstract