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*
Institute of Pathology,
Division of Neuropathology, and
Cancer Research Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106
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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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Earlier in situ hybridization and Northern blot studies revealed that in addition to the CNS, the mRNA of PrPC is expressed in many tissues (11, 12). Expression of PrPC mRNA is also regulated prenatally and postnatally (13, 14), suggesting that PrPC may be critical for development. However, it was found that mice devoid of PrP gene (PrPC-/-, PrPC knockout mice) were fertile and, in general, quite normal (15, 16). Although the expression of PrPC is obligatory for the pathogenesis of prion diseases in mice (17, 18, 19), the role that PrPC plays in normal cellular physiology is not clear. Accumulating evidence suggests that PrPC is a copper-binding protein (20, 21, 22, 23) and may play a role in modulating oxidative stress (24, 25, 26, 27).
The pathologic lesions associated with prion diseases are found exclusively in the CNS (5, 6). However, many studies indicate that non-CNS tissues also contribute to the pathogenesis of the disease (6). For example, the host lymphoid system is important in experimental models of prion diseases. In infected mice, PrPSc has been found in all components of the lymphoid-reticular system, including lymph nodes, spleen, and Peyer’s patches (28, 29, 30). Furthermore, in vivo activation of the immune system has been reported to decrease the incubation period after infection (31). Mitogen-activated murine lymphoid cells are 100-fold more susceptible to in vitro infection with PrPSc than nonactivated cells (32). Mice with SCID, which lack both T and B cells, do not support propagation of PrPSc in the spleen (33, 34). Follicular dendritic cells in the spleen have been reported to be essential for PrPSc infection (35, 36). The reason that follicular dendritic cells are uniquely susceptible to PrPSc is not known. To generate a better understanding of the role that the host immune system plays in the pathogenesis of prion diseases requires a detailed analysis of the expression of PrPC in the cells and tissues of the immune system. However, due to a lack of monoclonal reagents, little is known about the expression of PrPC in different immune compartments of the mouse.
The hemopoietic cell system is highly dynamic, with continual removal and renewal of all cell types (37, 38, 39). In adult mice, hemopoietic cell development occurs in the bone marrow. A single pluripotent hemopoietic stem cell (HSC) can give rise to essentially all the blood cells in the peripheral tissues (37, 38, 39). Accumulated evidence suggests that during hemopoietic differentiation, an HSC first commits itself either to become a common lymphoid progenitor (CLP) (40), which gives rise to all the T cells, B cells, and NK cells, or a common myeloid progenitor (CMP) (41), which gives rise to all myeloid cells, including erythroid cells, monocytes, granulocytes, and megakaryocytes. These early progenitor cells have been characterized and isolated by surface Ag expression. In most studies, sorting of these progenitor cells is based on the lack of a panel of lineage-specific marker (e.g., CD45RA/B220, CD2, CD4, CD8, Gr-1, CD11b/Mac-1, etc.). Some of the markers being used for the positive selection are Sca-1, c-kit, CD34, CD24/heat-stable Ag, CD43, and IL-7R, etc. CD43, a transmembrane cell surface sialomucin-type glycoprotein, is expressed on mouse HSC, CLP (40), and myeloid progenitor (Ly6+, Lin-, and CD43+) (42) as well as during early B cell differentiation (43). IL-7 has also been extensively studied for its critical role in the early stages of initiating lymphocytes commitment and maturation (44, 45). The IL-7R is present on CLP, but not on HSC and CMP (40, 41, 45).
The major purpose of this study is to document PrPC expression in peripheral and primary lymphoid organs of the mouse using a well-characterized anti-PrPC mAb that we have recently developed (46, 47). We found that most of the mature lymphocytes in the peripheral lymphoid tissues lack detectable PrPC. However, PrPC is readily detectable on the surface of immature thymocytes in the thymus and subpopulations of early progenitor cells as well as pro-B cells in the bone marrow. Therefore, during the maturation of T and B cells, the expression of PrPC is repressed and becomes restricted to only a small population of mature lymphocytes. Studying the expression of PrPC in hemopoietic cells may provide new insights into the pathogenesis of prion diseases, the physiological function of PrPC, and the differentiation of hemopoietic cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The methodology for the generation of anti-PrPC mAbs has been described in our previous work (46, 47). The anti-PrPC mAb, 8H4, used in this study is of the IgG1 subclass. The control mAb, GKW A3, is also an IgG1 Ab that is specific for human CD44. The original breeding pairs of 129/Ola PrPC-/- mice were kindly provided by Jean Manson (Edinburgh, U.K.). Wild-type 129/Ola mice (Harlan, Indianapolis, IN) were used as controls. C57BL/6.Rag-1-/- and wild-type C57BL/6 mice were kindly provided by P. V. Lehmann in our department.
Immunofluorescent staining and FACS analysis
The single cell suspension of bone marrow, thymus, or the spleen was prepared from age- and sex-matched PrP+/+ and PrP-/- mice. Cells were washed with a washing buffer (PBS supplemented with 5% newborn calf serum, 0.1% NaN3, pH 7.4) and blocked with Fc-Block (PharMingen, San Diego, CA) on ice for 25–30 min. Cells were then incubated with purified mAb 8H4 or an isotype-matched, control Ab on ice for 45 min. Cells were washed twice and incubated with FITC-conjugated F(ab')2 of goat anti-mouse IgG Fc-specific Ab (Chemicon, Temecula, CA) for 25 min on ice. Finally, samples were washed and analyzed immediately. For two- and three-color immunofluorescence staining, the protocol was similar to that described above in terms of the incubation and wash steps. All the reagents used and listed below were from PharMingen, except the anti-PrP mAb: Biotinylated 8H4-, PE-, or Cy-Chrome-conjugated streptavidin, PE anti-CD4, PE anti-CD8, PE or FITC anti-B220, PE anti-surface IgM (anti-sIgM), PE anti-CD43, FITC anti-Gr-1, rat anti-mouse IL-7R, and FITC-conjugated goat anti-rat IgG. Stained cells were analyzed immediately by FACScan (Becton Dickinson, San Jose, CA). At least 5000 cells were analyzed per gating in all experiments, and all experiments were repeated at least three times for consistency.
Immunohistochemical staining of tissues
Age- and sex-matched PrP+/+ and PrP-/- mice were first perfused with a fixation buffer (4% paraformaldehyde in 0.1 M phosphate buffer). The spleens were removed and immersed in fixation buffer for 5 h, and subsequently switched into a 18% sucrose/phosphate buffer at 4°C overnight. OCT-embedded frozen sections (7 µm) were prepared, air dried overnight, and stored at -80°C till use. For tissue staining, slides were first incubated in 0.1 M citrate buffer (pH 6) at 95°C for 30 min, and then blocked with 5–10% normal mouse serum. To reduce nonspecific background, we used a biotinylated anti-PrP mAb 8H4 and a biotinylated, irrelevant control mAb, GKW A3, that is specific for human CD44 to stain the sections. Stained sections were then developed with an ABC staining kit (Vector, Burlingame, CA). Visualization was achieved by using the diaminobenzidine system (Vector), as described by the manufacturer. Slides were eventually counterstained with hematoxylin.
Western blotting of tissues
Brain tissues, bone marrow cells, or the thymus obtained from PrP+/+ or PrP-/- mice were homogenized (brain and thymus) or lysed (bone marrow cells) with lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM Tris, pH 7.4, 2 mM PMSF). Defined amounts of protein from each tissue were loaded and separated in 15% polyacrylamide gel, then transferred to Immobilon P (Bio-Rad, Richmond, CA) for 2 h at 90 V. Membranes were incubated overnight at 4°C with anti-PrP mAb 8H4. Bound mAbs were detected with an HRP-conjugated F(ab')2 of goat anti-mouse IgG Fc-specific Ab (Chemicon). The blots were developed using an enhanced chemiluminescence system (Pierce, Rockford, IL), as described by the manufacturer. Prestained m.w. markers (Bio-Rad) were used as standards.
Stimulation of bone marrow cells with PMA
Single cell suspensions of the bone marrow from
PrP+/+ mice or PrP-/-
mice were prepared as described earlier. For the proliferation assay,
cells were cultured in vitro at 2.5 x 105
cells/well in 96-well plates in triplicate with complete medium: RPMI,
1% antibiotics, and 10% preselected FCS. PMA at 20 ng/ml was also
added into some of the wells. Plates were incubated in a 37°C
incubator with 5% CO2. After culturing for
48 h, cells were pulsed with 1 µCi of
[3H]thymidine for an additional 16 h.
Cells were harvested with an automatic harvester (Wallac, Gaithersburg,
MD), and the amount of radioactivity incorporated was determined by
scintillation counting (Wallac, 1205 
plates). For FACScan analysis,
10–15 x 106 cells were cultured with or
without PMA (20 ng/ml) in 25-cm2 flasks with
complete medium for 48 h, and then the cells were collected,
washed, and stained as described earlier. Only viable cells were gated
for analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first investigated the expression of PrPC
on the surface of lymphocytes in the spleen using mAb 8H4. The mAb 8H4
reacts with a conformational epitope, which is located between amino
acid residues 145 and 200 in the C-terminal region of
PrPC (46, 47). Representative
results of two-color flow cytometry analysis of the spleen cells from
wild-type 129/Ola and 129/Ola-PrP-/- mice are
shown in Fig. 1
A.
Approximately 10–20% of the spleen cells express
PrPC on their surface (right two
quadrants). Combined with T and B lymphocyte-specific markers,
10–15% of the CD4+ T cells, 5–7% of the
CD8+ T cells, and 10–15% of the
sIgM+B220+ mature B cells
are PrPC+ (Fig. 1). No significant numbers of
PrPC+ cells are detected in the spleen of
PrPC-/- mice (Fig. 1). Similar results were
obtained with lymph node cells (results not shown).
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B clearly demonstrated that intense staining of
PrPC was detected in the myofibroblasts in the
capsule of the spleen from PrPC+/+ mice (see
arrow in Fig. 1B-a). Most of the cells in the lymphoid
compartment, however, did not react with mAb 8H4, except the follicles
in some of germinal centers. Under higher magnification, some
lymphocyte-like cells in the follicles showed immunoreactivity, and it
appeared that the staining was not evenly distributed on the membrane
(see arrows in Fig. 1B-c). However, not all of the
lymphocytes were stained. Some network structures between cells also
reacted positively with the anti-PrP mAb (arrow in Fig. 1B-c). No immunoreactivity was detected in the spleen of
PrPC-/- mouse (Fig. 1B-b,
d). These results provide additional support for our
conclusion that most of the mature lymphoid cells in the spleen do not
express detectable PrPC. Because most of the peripheral mature T and B cells do not express PrPC, we therefore focused our studies on whether PrPC is expressed in immature T cells in the thymus or progenitor cells in the bone marrow, the sites of T and B cell development.
Most thymocytes are PrPC+, while the expression of PrPC in the bone marrow is heterogeneous and cell type dependent
The expression of PrPC in the thymus and
bone marrow cells was first demonstrated by immunoblotting of lysates
prepared from either wild-type PrPC+/+ mice or
PrPC-/- mice that were strain, age, and sex
matched with mAb 8H4. Brain lysates from PrPC+/+
mice were used as a positive control. PrPC in the
brain is present in three isoforms, which migrate as 39- to 42-kDa, 32-
to 37-kDa, and 27-kDa proteins (46) (Fig. 2A). The three isoforms of
PrPC also could be detected with mAb 8H4 in bone
marrow cells and the thymus from PrPC+/+ mice,
but not from PrPC-/- mice (Fig. 2A).
In general, the brain contains 
5 times more
PrPC than the bone marrow and thymus.
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B).
Because the majority of the thymocytes are immature T cells, these
results suggest that the expression of PrPC is
repressed during the development of T cells.
We next determined the expression of PrPC on the
surface of bone marrow cells by flow cytometry. Bone marrow cells can
be arbitrarily divided into four groups based on the size and cellular
granularity (Fig. 2C). Cells in region 1 (R1) are
predominantly small lymphocytes and progenitor cells; cells in region 2
(R2) are larger lymphocytes and maturing monocytes; cells in region 3
(R3) are myeloid cells, mainly maturing granulocytes; cells in region 4
(R4) are mostly erythroid cells and their precursors.
In terms of PrPC expression, cells in R1 can be
divided into two subpopulations: PrPC-positive
cells, which represent 45–60% (n = 5) of the
cells, and PrPC-negative cells. Cells in R2 can
also be divided into two groups; 30–40% of the cells express
moderate levels of PrPC, while the rest do not.
In contrast, most of the erythroid cells in R4 and maturing
granulocytes in R3 uniformly express moderate levels of
PrPC (Fig. 2C). The expression of
PrPC on the surface of RBC was confirmed by
direct staining of peripheral blood with mAb 8H4 (results not shown).
These results suggest that the expression of PrPC
in bone marrow cells is heterogeneous and cell type dependent.
PrPC is preferentially expressed on CD43+, B220-, sIgM-, and IL-7R- cells in the bone marrow
The morphology of the early progenitor cells is known to resemble
small lymphocytes (37). Therefore, we used two- and
three-color flow cytometry to further delineate the
PrPC-expressing cells in R1. Four
well-characterized differentiation markers were used in these studies:
1) CD45RA (B220), which is a B cell lineage-specific marker present on
all B cells of different stages; 2) sIgM, a marker for immature B cells
and mature B cells (48); 3) IL-7R 
-chain (IL-7R), a
marker for CLP cells, pro- and pre-B cells, as well as pro-T cells
(44, 49, 50); 4) CD43, a marker that is present in early
progenitor cells including HSC, CLP (40), myeloid
progenitors (Ly6+, Lin-,
and CD43+) (42), pro-B cells, pre-B
cells (low) (43, 51, 52), and maturing granulocytes (high)
(42), but not RBC (53).
Results from a representative experiment are shown in Fig. 3. We found that most of the
PrPC+ cells in R1 lack B220. Only 10.5% (range
10–15%, n = 5) of the cells express both B220 and
PrPC (Fig. 3A, up-right
panel). Based on B220 expression level, these
PrPC+ cells can be further divided into
PrPC+B220high and
PrPC+B220low populations.
Similar to earlier findings in the spleen, in the bone marrow the
majority of the PrPC+ cells also lack sIgM. Only
7.6% (range 5–10%, n = 5) of the cells are positive
for both PrPC and sIgM (Fig. 3A,
middle right panel). Many of the PrPC+
cells in the bone marrow also lack IL-7R (Fig. 3A,
bottom right panel). However, 17% of cells in region 1,
which represent 30% of the total
PrPC-expressing cells, do express IL-7R
(n = 5). In contrast, about one-half of the
IL-7R+ cells are PrPC-
(Fig. 3A, bottom right panel).
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B, middle right panel). Some
PrPC+ cells express neither CD43 nor B220. Only a
small percentage of
CD43+B220low and
CD43-B220high cells are
PrPC+ (Fig. 3B, middle right
panel). For the sIgM and B220 combination, most of the
PrPC+ cells are sIgM- and
B220- (Fig. 3B, bottom right
panel). A small percentage (6.2%) of
sIgM+B220low or high cells
representing immature and mature B cells are
PrPC+ (Fig. 3B, bottom right
panel), while the majority of these cells are
PrPC- (Fig. 3B, bottom left
panel). Approximately 15% of the
sIgM-B220low cells, which
contain pro- and pre-B cells, are PrPC+ (Fig. 3B, bottom right panels).
Collectively, these results suggest that 40–60% of the cells in R1
are positive for PrPC, and among these cells,
65% of them have the cell surface phenotype of
PrPC+, CD43+,
IL-7R-, B220-, and
sIgM-, a phenotype that correlates with the
early progenitor cells in bone marrow. Another population, which
accounts for 20% of the total PrPC+ cells,
has the phenotype of PrPC+,
CD43+/-, IL-7R+,
B220low/high, and sIgM+/-.
These cells may represent cells in different stages of B cell
differentiation, i.e., pro-, pre-, immature, and mature B cells.
However, the majority of these maturing B cells are
PrPC-, indicating that
PrPC is repressed during B cell development. A
small population of cells is PrPC+, but
CD43-B220- (Fig. 3B, middle right panel). They may represent some
erythroid cells and their precursors.
Most arrested pro-B cells in the bone marrow of Rag-1-/- mice express PrPC
Mature T and B cells are absent in mice lacking the recombinase gene, Rag-1 (54). Previous results suggested that PrPC might be preferentially expressed in early progenitor cells in the bone marrow; thus, we reasoned that more PrPC+B220- progenitor cells might accumulate in the bone marrow of Rag-1-/- mice. Moreover, because B cell development in Rag-1-/- mice is blocked at the pro-B cell to early pre-B cell stage (54), studying bone marrow cells in these mice may allow us to determine the pattern of PrPC expression specifically on pro-B cells.
First, we compared the expression of B220 and sIgM by two-color flow
cytometry analysis of bone marrow cells from
Rag-1-/- mice with that from wild-type B6 mice.
We found that there are no sIgM-positive cells; only
sIgM-B220low cells are
detectable in Rag-1-/- mice, confirming that B
cell differentiation in these mice has been interrupted (Fig. 4A). Second, consistent with
our hypothesis, bone marrow cells from Rag-1-/-
mice have more PrPC+B220-
cells (81%, range 75–85%, n = 3) than wild-type B6
mice (51%, range 45–60%, n = 3)
(p < 0.01) (Fig. 4B). Furthermore,
only B220low cells are present in
Rag-1-/- bone marrow, which correlates with
arrested pro-B cells. It is clear that almost all of the pro-B cells
are PrPC+ (Fig. 4B, right
panel). This
B220lowPrPC+ population of
cells is also positive for CD43 and IL-7R (data not shown).
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Stimulation of the PrPC+B220- progenitor cells with PMA
Our results suggested that most of the PrPC+ cells in the bone marrow are early progenitor cells. We next sought to determine whether these cells have the potential to differentiate further in vitro. We chose PMA rather than some other more specific growth modulators (i.e., IL-7), because many of the very early PrPC+ progenitor cells in bone marrow do not express IL-7R. Furthermore, PMA provides a much more potent and nondiscriminating signal than cytokines.
Bone marrow cells were cultured in medium alone or with PMA for 48
h. Subsequently, cells were collected, and the expression of
PrPC was determined by two-color flow cytometry.
After PMA stimulation, the number of cells in the
PrPC+B220- population is
decreased from 37.6% (range 30–45%, n = 3) to 14.3%
(range 11–20%, n = 3, p < 0.01)
(Fig. 5A, upper
panels), indicating that this population of cells contains
progenitor cells able to give rise to other types of cells, B cells in
this case. Furthermore, stimulation with PMA increased the number of
sIgM+B220high cells by
4-fold (from 11% to 46%, n = 3), and also increased
the corresponding number of
PrPC+B220+ cells from 7%
(range 5–8%, n = 3) to 16% (range 13–17%,
n = 3, p < 0.01) (Fig. 5A
boxes, bottom panels). The effect of PMA was also monitored
by quantifying the incorporation of
[3H]thymidine as an indicator of cellular
proliferation. Bone marrow cells proliferated much more in the presence
of PMA than cells cultured in medium alone (Fig. 5B).
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Cells in region 3 uniformly express PrPC
(Fig. 2C, R3). This population of cells has been shown to
express high level of CD43. We used an additional differentiation Ag,
Gr-1, which is specifically expressed on developing granulocytes
(55, 56) to verify the phenotype of the
PrPC+ cells in region 3. Two-color flow cytometry
analysis with mAb 8H4 and anti-Gr-1 mAb revealed that most of the
cells in region 3 express very high levels of Gr-1 (Fig. 6). More importantly, the majority of the
CD43+ and Gr-1+ cells are
also PrPC+ (Fig. 6). These results provide direct
evidence that most of the maturing granulocytes in the bone marrow
express PrPC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). These findings are further
supported by immunohistochemical staining of the spleen. The
immunoreactivity in the spleen is mainly associated with the splenic
capsule. Most of the cells in the lymphoid compartment do not react
with the anti-PrP mAb. However, some cells in the follicles of
germinal centers in the spleen do react positively with the mAb. The
follicles of germinal centers are home for many cell types, including B
cells, T cells, monocytes, and FDCs. Therefore, the small population of
PrPC+ T and B cells detected by FACScan analysis
may represent the cells in the germinal centers. However,
immunohistochemical staining does not provide sufficient resolution to
positively identify the nature of these PrPC+
cells. Experiments are now in progress to use immunoelectron microscopy
to directly identify the PrPC+ cells in the
spleen.
In contrast to mature lymphoid cells, most thymocytes are
PrPC+, and some subpopulations of bone marrow
cells are also PrPC+ (Fig. 2). These results
provide strong evidence that the expression of
PrPC is regulated during T and B lymphocyte
differentiation. In the current study, we have focused our attention on
the development of hemopoietic cells in the bone marrow because bone
marrow is the primary site in which all hemopoietic progenitor cells
are generated in adult mice.
In the bone marrow, erythroid cells and maturing granulocytes uniformly
express PrPC. In contrast,
PrPC expression levels in other bone marrow cells
are heterogeneous and cell type dependent (Fig. 2). Approximately
50–60% of the cells in region 1 express PrPC.
Most of the PrPC+ cells are
CD43+, but IL-7R-,
B220-, and sIgM- (Fig. 3). These cells also lack lineage-specific markers such as Thy-1, CD19,
Mac-1, CD3, or Gr-1 (not shown), suggesting that these cells might be
early, uncommitted progenitor cells.
B220 is one of the earliest markers expressed on committed B cells
(48). To incorporate our findings into a general scheme of
bone marrow cell differentiation, we, therefore, subdivided
PrPC+ cells in region 1 into three subclasses
based on their B220 expression levels: B220-
(box I), B220low (box II), and
B220high (box III) populations (Fig. 7).
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40–60% of the total R1 cells, the majority
of cells are PrPC+, and most of these
PrPC+ cells are
B220-CD43+ (Fig. 3B). Bone marrow progenitor cells known to express CD43, but
lacking B220, include HSC cells, CLP (40), and myeloid
progenitors (Ly6+, Lin-,
and CD43+) (42). The identities of
these cells may be further defined based on the expression of IL-7R.
IL-7R is present on CLP, but not on HSC or CMP (40, 41, 45). We found that 25–35% of cells in R1 are
IL-7R+, and at least one-half of the
IL-7R+ cells are PrPC+
(Fig. 3A). It is possible that some of these
PrPC+IL-7R+ cells may
represent CLP. However, it is unlikely that the numbers of HSC, CLP,
and myeloid progenitor cells can account for all the
PrPC+B220-CD43+
cells, which represent 40% of the cells in R1. Therefore, there may
be additional populations of
PrPC+B220-CD43+
cells with unknown identities. Thymic stem cells have been reported to
be present in mouse bone marrow (57). Some of the
PrPC+B220- cells we have
identified in the bone marrow may be thymic stem cells that are
designated to migrate to the thymus. This speculation is supported
indirectly by our finding that most thymocytes are
PrPC+. Other candidates include NK cells and
their precursors (58) or other intermediate cell types at
different stages of differentiation, which are
CD43+B220-.
The interpretation that PrPC may be
preferentially expressed on progenitor cells is further supported by
our PMA stimulation experiment in vitro (Fig. 5). Stimulation of bone
marrow cells with PMA significantly decreased the number of
PrPC+B220- cells from 38%
to 14%, suggesting that PMA be able to stimulate the potential
PrPC+B220- progenitor
cells to give rise to downstream cell types, which resulted in the
increased B220+ cells. However, we cannot rule
out the possibility that PMA might have preferentially stimulated the
proliferation of PrPC+B220+
cells rather than inducing the differentiation of
PrPC+B220- cells.
We have provided evidence that PrPC is preferentially expressed on a subpopulation of not yet committed, progenitor cells in the bone marrow. However, the precise identities of these cells remain to be determined. Additional in vitro cell sorting and culturing experiments as well as in vivo reconstitution experiments will be required to directly reveal the fate of these PrPC+ cells. Further investigation of PrPC expression in combination with additional surface markers and cytoplasmic markers by FACS and transcription factors by PCR should further delineate the developmental potentials of these cells.
The second group is B220low cells in box II.
Developing B cells that are known to express low levels of B220
include: pro-B cells
(CD43+IL-7R+sIgM-),
pre-B cells
(CD43+/-IL-7R+sIgM-),
and immature B cells
(CD43-IL-7R-sIgM+).
In wild-type mice, a small fraction of the
B220low cells is PrPC+
(Fig. 3A); therefore, most of the cells that have already
committed to the B cell lineage do not express
PrPC. Interestingly, we found that the
B220low cell is the only
B220+ population detected in
Rag-1-/- mice, and most, if not all, of these
cells are PrPC+. Because B cell development is
blocked at the transition from pro-B cells to pre-B cell stage in
Rag-1-/- mice (54), the
accumulation of developmentally arrested pro-B cells and earlier
precursor cells in these mice may account for the
B220lowPrPC+ cells in the
bone marrow of Rag-1-/- mice. These results
suggest that maturing B cells before pre-B stage express
PrPC. Furthermore, the
B220lowPrPC- population,
which accounts for 70–80% of the total B220low
cells in wild-type mice, is undetectable in
Rag-1-/- mice (Fig. 4), suggesting that
recombination events may influence the transition of cells from being
PrPC+B220low to
PrPC-B220low. The
mechanism(s) by which the Rag-1 gene influences this process
is not known. We observed that 15% pre-B and immature B cells are
PrPC+, respectively, while the majority of them
are PrPC-. Because pre-B and pro-B express
IL-7R, most of the IL-7R-positive cells seen in Fig. 3A
(bottom right panel) could be pre-B and pro-B cells, while
the PrPC+IL-7R+
double-positive population may represent pro-B cells. Collectively,
these results further suggest that B cell differentiation is
accompanied by the down-modulation of PrPC
expression through the pre-B cell stage. We did not study thymocytes in
Rag-1-/- mice because of difficulties in
obtaining the thymus from Rag-1-/- mice,
resulting from the atrophy of the thymus in these mice.
Mature B cells with high levels of B220, which account for 10–20% of R1 cells, are the main population in box III. Only a small population, 15–25%, of B220+sIgM+ mature B cells in box III expresses PrPC. Our observations that most of the mature B cells in the bone marrow do not express PrPC are consistent with our finding in the peripheral tissues by flow cytometry analysis and immunohistochemical staining. The reason that only a small number of mature B cells maintain expression of PrPC while the majority of them do not along B cell maturation is not clear. Additional in vivo and in vitro studies will be required to verify whether PrPC is preferentially expressed on functional subsets of B cells (e.g., CD5+ B cells, memory B cells, or Ab-producing B cells).
The current study indicates that PrPC expression is regulated during lymphocyte development in the bone marrow and thymus. However, the exact role PrPC plays in hemopoietic cell differentiation and lymphocyte function is not clear. Other investigators have reported that there was a significant reduction of Con A-induced T lymphocyte proliferation in PrP-/- mice compared with wild-type mice (59). However, using PrP-/- mice in two different genetic backgrounds, we found that T lymphocytes from PrP-/- mice appeared to respond normally to Con A, and to immobilized anti-CD3 mAb (results not shown). The reasons for these discrepancies are not known. Furthermore, based on our immunohistochemistry study and flow cytometry analysis, the lymphoid compartments in PrP-/- mice appeared to be normal, and there was no significant difference related to the hemopoietic development in the bone marrow of wild-type and PrP-/- mice. Moreover, both wild-type and PrP-/- mice have comparable levels of all classes of serum Ig (T. Liu, unpublished results). Therefore, it is likely that PrPC only plays a subtle role in the development or function of the murine lymphoid system. Alternatively, the function of PrPC may be compensated by another gene product in PrP-/- mice. The significance of our findings with regard to the pathogenesis of prion disease in mice is also unclear. Our findings that most of the T and B cells in peripheral lymphoid organs lack PrPC are in good accordance with previous studies that indicate mature T and the PrPC expression of B cell are not essential for PrPSc infection (35, 60, 61). However, it remains to be determined whether early progenitor cells in the bone marrow play a role in PrPSc infection.
In summary, we have provided evidence that PrPC is expressed on subpopulations of early progenitor cells in the bone marrow and immature thymocytes. Further studies on the expression of PrPC in bone marrow progenitor cells may facilitate the identification of additional subsets of bone marrow progenitor cells. More importantly, the precise documentation of PrPC expression on the development of hemopoietic cells may provide new insights into the normal physiologic functions of PrPC and the pathogenesis of prion diseases.
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2 Abbreviations used in this paper: PrPC, normal cellular prion protein; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; HSC, hemopoietic stem cell; sIgM, surface IgM.
Received for publication September 27, 2001. Accepted for publication January 16, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helices into -sheets features in the formation of the scrapie prion proteins. Proc. Natl. Acad. Sci. USA 90:10962. actin and prion protein mRNAs during brain development in mouse. Brain Res. Mol. Brain Res. 10:343.[Medline]
chain (CD25, TAC) expression defines a crucial stage in pre-B cell development. Int. Immunol. 6:1257.
5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice. Cell 77:133.[Medline]
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