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Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1556-1561
Prion Protein Expression in Human Leukocyte Differentiation
By
Vincent C. Dodelet and
Neil R. Cashman
From the Department of Microbiology and Immunology, the Department of
Neurology and Neurosurgery, Montreal Neurological Institute and
Hospital, McGill University, Montreal, Québec, Canada.
 |
ABSTRACT |
The cellular isoform of the prion protein (PrPC) is a
small glycoprotein attached to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol anchor. This molecule is involved in
the pathogenesis of prion diseases in both humans and animals. We have
characterized the expression patterns of PrPC during human
leukocyte maturation by flow cytometry with monoclonal antibodies to
PrPC, the glycan moiety CD15, and the stem cell marker
CD34. We observe that prion protein is present on CD34+
bone marrow (BM) stem cells. Although lymphocytes and monocytes maintain PrPC expression throughout their differentiation,
PrPC is downregulated upon differentiation along the
granulocyte lineage. In vitro retinoic acid-induced differentiation of
the premyeloid line HL-60 into granulocyte-like cells mimics the
suppression of PrPC in granulocyte differentiation, as both
PrPC mRNA and protein are downregulated. These data suggest
that selected BM cells and peripheral mononuclear cells may support
prion agent replication, because this process is dependent on
availability of PrPC. Additionally, retinoic acid-induced
extinction of PrPC expression in HL-60 cells provides a
potential model to study PrP gene regulation and protein function.
Finally, these data suggest the existence of cell-specific glycoforms
of PrPC that may determine cellular susceptibility to
infection by the prion agent.
| |
INTRODUCTION |
THE PRION PROTEIN is best known for its
involvement in the transmissible spongiform encephalopathies (TSE) or
prion diseases, a group of human and animal neurodegenerative diseases
which include Creutzfeldt-Jakob disease in humans, scrapie in sheep and
goats, and bovine spongiform encephalopathy in cattle. The pathology of
the TSEs is characterized by neuronal loss, spongiform change, gliosis,
and accumulation of an abnormal protease-resistant protein designated
PrPSc in both brain and peripheral tissues, including
organs of the lymphoreticular system. The prevalent
"protein-only" hypothesis of prion disease proposes that
PrPSc is indeed the infectious agent, and that this
modified, pathogenic form of the normal cellular protein replicates by
converting the normal isoform to the disease isoform.1
The normal cellular isoform of the prion protein PrPC is a
small glycosylphosphatidylinositol (GPI)-anchored cell-surface protein expressed predominantly in the brain, but also in a wide variety of
peripheral tissues, including peripheral mononuclear blood cells.2,3 PrPC is present in all vertebrates
examined to date, is widely expressed during embryogenesis, and has
been strongly conserved throughout evolution, suggesting a vital
function for the protein.4-6 Surprisingly, PrP null mice
have shown that the protein is apparently not necessary for normal
murine development or mature survival because these mice are viable, do
not show any obvious developmental phenotype, and show no behavioral
abnormalities up to 93 weeks of age.7,8 Ablation of the
prion gene renders these mice resistant to experimental scrapie and
obviates accumulation of PrPSc.8 More
target-directed assays have shown subtle abnormalities in null mice
such as defective GABAA receptor-mediated fast inhibition and impaired long-term potentiation, altered circadian rhythms, and
loss of cerebellar Purkinje cells.9-11 These phenotypes are reminiscent of certain features of TSEs and suggest that loss of the
normal function of this protein may contribute to
disease.12
Clues to the function of PrPC may be gleaned by examination
of cell-specific expression patterns. In peripheral blood of humans and
rodents, PrPC is detected at the cell surface of
lymphocytes and monocytes, but is absent from erythrocytes and
granulocytes.2,3 Our laboratory has previously determined
that PrPC expressed by mononuclear cells may play a role in
cell activation.2 In this study, we further characterized
the expression patterns and gene regulation of the protein in cells of
the immune system. We now show that PrPC is expressed very
early in hematopoiesis, indicated by its presence on CD34+
stem cells. Differentiation along the lymphocyte or monocyte lineages
supports the expression of surface PrPC, while
differentiation along the granulocyte lineage progresses with
concomitant downregulation of PrPC surface protein. This
cell-type specific extinction of PrPC in granulocyte
differentiation can be recapitulated in vitro by retinoic acid
induction of HL-60 cells.
These results allow us to better define the regulation of the
expression of PrPC in human leukocytes, which has potential
implications for human and animal prion diseases. In addition, our
studies provide a novel in vitro system with which to study PrP gene
regulation and protein function.
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MATERIALS AND METHODS |
Cells.
Human peripheral blood leukocytes were isolated from whole blood from
healthy volunteer donors and separated using Lymphocyte-poly (Cedarlane, Hornby, Ontario, Canada) density centrifugation. The two
resulting layers were pooled, washed three times with
phosphate-buffered saline (PBS), and stained for flow cytometry
analysis as described in the following section.
Human low-density bone marrow (BM) cells were isolated by Ficoll-Paque
(Pharmacia, Baie D'Urfé, Québec, Canada)
density centrifugation of BM aspirates from patients undergoing
diagnostic BM evaluation (kindly provided by Dr Alan Brox, Royal
Victoria Hospital, McGill University). Cells were washed extensively
and stained for flow cytometry analysis.
The human premyeloid line HL-60 (ATCC CCL-240) was maintained in RPMI
1640 medium (GIBCO-BRL, Burlington, Ontario, Canada) supplemented with
10% fetal bovine serum (FBS), 2.5 mg/mL penicillin, 2.5 mg/mL
streptomycin, and 2 mmol/L glutamine. Differentiation along the
monocytoid lineage was induced by culture in 100 nmol/L of phorbol
12-myristate 13-acetate (PMA; Sigma, St Louis, MO) for 3 days.
Differentiation along the granulocyte lineage was induced by culture in
1 mol/L all-trans retinoic acid (Sigma) for 0, 1, 3, and 6 days. Cells were then stained for flow cytometry analysis or procured
for isolation of total RNA.
Monoclonal antibodies (MoAbs) and flow cytometry.
The MoAb 3F4, which uniquely recognizes PrPC at the cell
surface2,13 (and N.R.C., unpublished data),
was prepared as ascites fluid in pristane-primed Balb/c mice, and used
unconjugated at a 1:5,000 dilution. 3F4 was followed by either a goat
anti-mouse Fab fluorescein isothiocyanate (FITC) conjugate or a donkey
anti-mouse phycoerhythrin (PE) conjugate (both from Jackson
Immunoresearch, West Grove, PA). Anti-CD15-FITC and anti-CD34-PE MoAb
direct conjugates were obtained from Becton Dickinson (Saint-Laurent,
Québec, Canada). FITC-conjugated, PE-conjugated, and
unconjugated isotype-matched irrelevant control MoAbs were included in
all experiments (Becton Dickinson and Sigma).
For immunofluorescence staining, cells were first incubated on ice for
30 minutes in PBS supplemented with 10% normal goat serum to block
nonspecific binding. Cells were then stained with the appropriate
MoAbs, each for 30 minutes on ice. Samples were analyzed on a FACSCAN
flow cytometer using LYSYS II software (Becton Dickinson).
Semiquantitative polymerase chain reaction (PCR) analysis.
HL-60 cells were differentiated with all-trans retinoic acid as
described. Cells were harvested at days 0, 1, and 3 and total RNA was
isolated using TRIzol (GIBCO-BRL). Total RNA was then treated with RQ1
RNase free DNase (Promega, Madison, WI) and re-extracted twice with
phenol-chloroform. Total RNA was reverse-transcribed to cDNA using
Superscript reverse transcriptase (GIBCO-BRL) according to the
manufacturer's instructions. This cDNA was then used for both prion
and  -actin amplification by PCR. Primers used for the PCR reactions
had the following sequences: human prion forward 5 -AAGCCTGGAGGATGGAACACT-3, reverse
5-GTTGCTGTACTCATCCATGGG-3, and -actin forward
5-ATGCCATCCTGCGTCTGGACCTGGC-3, reverse
5-AGCATTTGCGGTGCACGATGGAGGG-3. The primers for human
prion and -actin were designed to generate fragments of 434 and 606 bp, respectively. Two hundred nanograms of cDNA was added to the
reaction mixture containing PCR buffer, 0.5 mmol/L dNTPs (Pharmacia),
50 pmol of either primer set, and 0.5 µL Taq polymerase (GIBCO).
Samples were placed in a thermocycler (Perkin Elmer Cetus Corp,
Norwalk, CT) for 25 cycles of 94°C for 1 minute, 60°C for 2 minutes, and 72°C for 3 minutes. The PCR products were separated on
a 1% 1× Tris-acetate EDTA (TAE) agarose gel,
transferred to a nylon membrane overnight by capillary action, and the
Southern blots were probed with the appropriate random primed
[32]P-labeled probes for 16 hours at 42°C in
Rapid-Hyb buffer (Amersham, Burlington, Ontario, Canada).
Blots were washed, and exposed to Phosphor Screen and analyzed on a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
| |
RESULTS |
Prion protein expression in human peripheral blood cells.
Human peripheral blood leukocytes were analyzed for PrPC
surface immunoreactivity by flow cytometry
(Fig 1). Three main populations can be
defined on the basis of size (forward scatter [FSC]) and granularity
(side scatter [SSC]). When these three populations are
examined for PrPC and CD15 immunoreactivity, each presents
a unique staining profile. CD15 provides a convenient secondary marker
because of its differential distribution among
leukocytes.14-16 Lymphocytes have low to intermediate levels of PrPC and are CD15 , monocytes
are PrPC high and CD15 low, and granulocytes are
PrPC and CD15 high. Erythrocytes are negative for
both markers (data not shown). The absence of PrPC on
granulocytes suggests at least two possibilities: lymphocytes and
monocytes acquire PrPC during differentiation, or
granulocytes lose PrPC as they mature.
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| Fig 1.
Expression of prion protein versus CD15 by human
peripheral blood leukocytes. Leukocytes were isolated from whole blood
of normal donors, washed, stained for PrPC and CD15, and
analyzed by flow cytometry. A typical light scatter contour plot of
normal peripheral human blood mononuclear cells is shown in the upper
left. Analysis gates R1-R3 are set on forward and side scatter alone
and define three major subpopulations: lymphocytes (R1), monocytes
(R2), and granulocytes (R3). Contour plots of surface prion protein
versus CD15 for each of the three subpopulations are shown. Lymphocytes
are (PrP+, CD15), monocytes are
(PrP+, CD15lo-med), and granulocytes are
(PrP, CD15hi). The small amount of
PrP+ cells in panel R3 are due to contaminating
monocytes.
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Prion protein expression in human BM cells.
To examine the question of acquisition or suppression of
PrPC in leukocyte differentiation, we first examined
PrPC expression in CD34+ multipotential stem
cells. As shown in Fig 2, cells that
express CD34 coexpress surface prion protein. This suggests that prion protein may indeed be specifically downregulated in the granulocyte and
erythroid lineage, and that other lineages maintain their expression of
prion protein. We further investigated this possibility by analyzing
low density human BM cells by flow cytometry
(Fig 3). Cells that are differentiating
along the granulocyte lineage show a dramatic increase in CD15 staining
in the maturation process. These highly granular cells show a
progressive loss of PrPC surface immunoreactivity as they
gain CD15 staining. These data demonstrate that prion protein is
present on pluripotential stem cells, and that it is downregulated
during subsequent differentiation along the granulocyte lineage.
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| Fig 2.
Surface prion protein expression by human
CD34+ BM cells. Human BM aspirates were fractionated for
low-density BM cells to enrich for multipotential stem cells. Cells
were stained for CD34 and PrPC and analyzed by flow
cytometry. Contour plot of low-density human BM cells shows that all
CD34+ cells also express PrPC. Data were
analyzed using gates for both high side scatter and CD34 expression.
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| Fig 3.
Downregulation of surface prion protein during in vivo
granulocyte maturation. Human BM aspirates were fractionated for
low-density BM cells as described. Cells were washed and stained for
flow cytometry analysis as described in the legend to Fig 1.
Differentiating granulocytes displaying high side scatter were gated as
shown on left. As these cells mature along the granulocyte lineage and become CD15hi, there is a progressive loss of surface
PrPC staining.
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Regulation of prion protein expression in differentiating HL-60
cells.
To provide a more convenient model of myeloid cellular differentiation,
we examined the premyeloid cell line HL-60, which can be induced to
differentiate along either the granulocyte or monocyte lineages by
retinoic acid or phorbol esters, respectively.17-20 HL-60
cells express PrPC, which persists upon phorbol ester
induced differentiation into macrophage-like cells
(Fig 4). Conversely, induction of HL-60 cells into granulocyte differentiation by retinoic acid was associated with progressive downregulation of surface PrPC
(Fig 5). The cells were observed over a
6-day period, with the proportionally largest decline in surface prion
protein immunoreactivity occurring after the first 24 hours (Fig 5B).
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| Fig 4.
Surface prion protein expression by HL-60 cells persists
upon induction of monocyte lineage differentiation. HL-60 cells were cultured in the presence or absence of 100 nmol/L PMA for
3 days, then stained for flow cytometry analysis with MoAb 3F4.
Histograms show PrPC surface immunoreactivity relative to
an IgG2b isotype control. Minimal change is observed in
PrPC staining intensity between undifferentiated (A) and
differentiated cells (B).
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| Fig 5.
Downregulation of surface PrPC in retinoic
acid-induced granulocytic differentiation of HL-60 cells. HL-60 cells
were cultured in the presence of 1 mol/L all-trans retinoic
acid for 0, 1, 3, and 6 days, then stained for flow cytometry analysis
with MoAb 3F4. Basal level of PrPC staining in
undifferentiated cells (solid line) is shown relative to an IgG2b
isotype control (in gray) (A). The progressive decrease of
PrPC staining during differentiation (in black) is shown
relative to basal level (B through D).
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To investigate the role of gene transcription in the loss of
granulocyte PrPC immunoreactivity, we examined the level of
mRNA for the prion protein by semiquantitative PCR. To exclude
potential contamination by PrP genomic sequences, the total RNA extract
was treated with RNase-free DNase before PCR amplification. HL-60 cells
were differentiated with retinoic acid as described and harvested for
RNA extraction after 24 and 72 hours. A progressive decrease in the
level of prion mRNA can be observed, while the level of -actin mRNA
remains approximately constant (Fig 6).
This shows a strong association between decreased surface
PrPC immunoreactivity and decreased PrP mRNA, most probably
the result of decreased transcription triggered by retinoic acid
treatment.
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| Fig 6.
Downregulation of PrP mRNA in retinoic acid-induced
differentiation of HL-60 cells. HL-60 cells were cultured in the
presence of 1 mol/L all-trans retinoic and obtained at days 0, 1, and 3. Total RNA was isolated and reverse-transcribed to cDNA, which was then used for both prion and -actin amplification by PCR. The
PCR products were then Southern blotted with radioactively labeled
PrP and -actin probes. Blots were analyzed by PhosphorImager scanning.
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| |
DISCUSSION |
The normal cellular isoform of the prion protein, PrPC, is
highly expressed in brain as well as many peripheral tissues, including peripheral blood mononuclear cells. We have found that PrPC
is expressed by CD34+ multipotential stem cells in human
BM. PrPC is downregulated in stem cell differentiation to
the granulocyte lineage, but is maintained in lymphoid and monocyte
lineages. The downregulation of PrPC in myeloid
differentiation, at both the protein and mRNA levels, was modeled in
vitro by the induction of HL-60 cells to granulocytoid lineage by
retinoic acid. Our findings differ from Diomede et al,21
who reported PrPC expression in neutrophils, as well as
lymphocytes and monocytes. However, these studies were based on cell
populations of 95% purity, in which contamination by mononuclear cells
may have contributed to the PrP mRNA and protein detected by PCR and
immunoprecipitation, respectively. It is also at least formally
possible that PrPC is a cell-surface molecule in
mononuclear cells detectable by flow cytometry, and an internally
sequestered protein in neutrophils.
The regulation of expression of PrPC is incompletely
understood. Despite the tissue-regulated and developmentally regulated expression of PrPC, the immediate 5 region of the
transcriptional start site does not contain a TATA box, but displays a
GC-rich sequence similar to that observed in many housekeeping
genes.22 Enhancer elements have been mapped upstream of the
start site, and are also apparently present in the long first intron
(~10 kb) detected in all vertebrate species whose genomic
PrPC organization is known.23,24 Our study
suggests that retinoic acid-responsive elements participate in the
cell-specific regulation of PrPC, and provides a
biologically relevant in vitro paradigm to investigate the molecular
basis of this regulation. Alternately, retinoic acid may be inducing
the expression of trans-acting proteins that act at a silencing
element controlling PrP expression. The regulation of prion protein
expression in granulocytes and erythrocytes may underlie a general
regulatory mechanism that could be exploited to reduce the amount of
PrPC available for conversion in the prion diseases,
because it has been shown that endogenous expression of PrP is
necessary for continued propagation of the scrapie agent.8
Prion infectivity apparently copurifies with PrPSc, the
protease resistant isoform of PrPC.25,26
Cell-free conversion of PrPC to PrPSc has now
been accomplished in vitro.27 Replication of the prion agent appears to be strictly dependent on availability of
PrPC, because mice homozygous for the targeted disruption
of the PrP gene are completely resistant to scrapie, and heterozygous
mice display a longer incubation and slower disease progression than wild-type mice.8 Thus, the distribution of PrPC
in hematogenous cells may predict the capability of that cell to
propagate and/or transmit prion infectivity in hosts transfused or transplanted with those cells. On the basis of our current report,
BM cells, including CD34+ stem cells, might be expected to
harbor the prion agent, despite current guidelines which do not
recognize BM as high-risk tissue.28,29 In the periphery,
monocytes and lymphocytes, which express PrPC, would be
expected to support prion replication, whereas PrPC
negative erythrocytes and granulocytes would not. This contention is
supported by a limited number of transmission
experiments30,31 and by recent data from Blattler et
al,32 who show that prion agent infectivity detected in
spleen by bioassay is dependent on the expression of PrPC
by spleen cells and does not accumulate by nonspecific
"carry-over" from the original innoculum.
An additional implication of our study is that PrPC must be
differentially glycosylated in the nervous system as opposed to the
periphery. PrPSc, the abnormal disease-associated isoform
of PrPC, has been found to possess a high proportion of
N-linked glycan chains terminating in the glycosyl moiety
stage-specific embryonic antigen 1 (SSEA-1), also known as
CD15.33 This carbohydrate moiety has been found to
participate in cell-cell adhesion and tissue differentiation early in
development, and probably plays an important role in immunocyte
adhesion in inflammatory processes.34,35 In the peripheral
blood, granulocytes, which express no PrPC, display
prominent immunoreactivity for CD15. Lymphocytes, which display no
surface CD15, do express PrPC. Only peripheral monocytes
possess surface immunoreactivity for both PrPC and CD15,
allowing the possibility that monocyte PrPC may be modified
with the SSEA-1 moiety. Because glycosylation may participate in the
brain-region-specific replication of the scrapie agent,36
it is possible that certain PrPC on different peripheral
cells might be more likely to participate in scrapie infection. Of
interest, some data indicate that the monocyte-macrophage cell lineage
is critical to the propagation of scrapie infectivity in the periphery.
Scrapie infection in severe combined immunodeficient mice is apparently
dependent on dendritic cells,37 which are tissue
macrophages specialized for immune presentation. Moreover, peripheral
blood cells containing scrapie infectivity are relatively radiation
insensitive,38 which is consistent with the end-mitotic
nature of peripheral monocytes. Finally, monocytes may be unique among
peripheral blood cells for their capability of processing
PrPC to PrPSc.39 Because other
peripheral blood cells also express PrPC, the capability of
monocytoid cells to support scrapie agent replication may relate to a
posttranslational modification, such as SSEA-1 modification of the
terminal PrPC glycans.
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FOOTNOTES |
Submitted July 3, 1997;
accepted October 24, 1997.
Supported by the Medical Research Council (MRC) of Canada Grant No.
MT12214, National Institutes of Health Grant No. AG09694, and Advanced
Bioconcept. V.C.D. was supported by PhD studentship grants from MRC and
Fonds de la recherche en santé du Québec.
Address reprint requests to Neil R. Cashman, MD, Montreal
Neurological Institute and Hospital, 3801 University, Montreal, Québec, Canada H3A-2B4.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge Dr Josephine Nalbantoglu for helpful
advice and discussion, and Dr Michelle Krakowski for critical reading
and inspiration.
| |
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Abstract
Introduction
Abstract