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Developmental Immunology, 2001, Vol. 8(3-4), pp. 291-304
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(C) 2001
Studies on Prion Replication in Spleen
ALEX J. RAEBERa*, FABIO MONTRASIO a, IVAN HEGYIa, RICO FRIGGa, MICHAEL A. KLEINa,
ADRIANO AGUZZIa and CHARLES WEISSMANNb?
alnstitute of Neuropathology, University Hospital, 8091 Ziirich and blnstitute of Molecular Biology, University of Ziirich, 8057 Ziirich,
Switzerland
Some of the early events following scrapie infection take place in the lymphoreticular system
(LRS) and result in significant replication of prions in lymphoid organs. The identity of the
cells in the LRS that produce prions and their role in neuroinvasion are still unknown. We
find that in the spleen of scrapie-infected mice, prions are associated with T and B cells and to
a somewhat lesser degree with the stroma, which contains the follicular dendritic cells
(FDC’s); curiously, no infectivity was found in lymphocytes from blood of the same mice.
Thus, splenic lymphocytes either replicate prions or acquire them from another source. Studies on PrP knockout mice with ectopic expression of PrP restricted to only B or T lymphocytes suggest that neither of these by themselves are competent for prion replication. To
determine whether B and T cells are able to pick up prions from other sources, irradiated
wild-type mice were reconstituted with PrP-deficient lymphohaematopoietic stem cells. Following intraperitoneal inoculation of these mice, no infectivity was found on splenic lymphocytes whereas the stroma (comprising the radiation-resistant, PrP-expressing FDC’s)
contained prions. These results imply that splenic lymphocytes can acquire prions, possibly
from FDC’s, but only if they express PrP.
Keywords: FDC, lymphoreticular system, prion propagation, scrapie, transgenic mice
INTRODUCTION
to be the main gateway for the agent in sheep scrapie,
kuru, BSE (Anderson et al., 1996; Wilesmith et al.,
1992) and likely new variant form of Creutzfeldt-Jakob disease (vCJD) (Will et al., 1996).
Peripheral infection, e.g. intraperitoneal (i.p.) infection into laboratory rodents likely mimics the natural
infection process more closely than i.c. inoculation.
The major clinical and pathological manifestations of
prion diseases are found in the central nervous system. Experimentally, the agent is most efficiently
transmitted by intracerebral (i.c.) inoculation into the
host. However the natural route of infection is more
likely via the gastrointestinal tract, which is believed
-
* Corresponding author: A.J. Raeber, Cytos Biotechnology AG, Wagistr. 21, 8952 Ziirich-Schlieren, Phone: (+411) 733 4032, Fax: (+411)
733 4019, e-mail: raeber@cytos.com
Present Address: MRC Prion Unit/Neurogenetics, Imperial College School of Medicine at St Mary’s, Norfolk Place, London, W2 1PG,
UK
291
292
ALEX J. RAEBER et al.
HOST CELLS IN THE LYMPHORETICULAR
SYSTEM INVOLVED IN PRION
PROPAGATION
Some of the initial events following scrapie infection
take place in the lymphoreticular system (LRS) and
result in significant replication of prions in lymphoid
organs, prior to neuroinvasion and replication of the
agent in the brain (Btieler et al., 1993; Eklund et al.,
1967; Fraser and Dickinson, 1970). It is noteworthy
that the host fails to mount a classical immune
response to the scrapie agent (Kasper et al., 1982),
presumably because it consists largely or entirely of a
host-derived protein. It is not known which cell types
in the lymphoreticular system are targets for the
scrapie agent and which, if any, mediate its transport
to neural sites of replication.
Studies involving splenectomy (Fraser and Dickinson, 1978), whole body irradiation (Fraser and Farquhat, 1987) and spleen fractionation (Clarke and
Kimberlin, 1984) suggested that mitotically quiescent
cells located in the stromal fraction of the spleen are
involved in propagation of the scrapie agent. It was
shown by immunohistochemistry that the protease-resistant isoform of PrP colocalizes with follicular dendritic cells (FDCs) in the spleen of
prion-infected mice (Kitamoto et al., 1991; Klein et
al., 1998; McBride et al., 1992).
Transport of prions from the periphery to the CNS
depends on elements of the LRS, as evidenced by the
fact that development of CNS disease after i.p. inoculation with scrapie is impaired or abolished in mice
with various forms of immunodeficiency, such as
SCID, RAG -/- or tMT mice, while i.c. inoculation
continues to be fully effective (Brown et al., 1997;
Fraser et al., 1996; Kitamoto et al., 1991; Klein et al.,
1997; Lasmezas et al., 1996). Although these studies
strongly suggest an important role for lymphoid cells
in the spread of the disease from peripheral sites to
the brain, there is also evidence for a direct neural
spread of the disease from the periphery to the CNS
albeit with a lower efficiency (Beekes et al., 1996;
Beekes et al., 1998; Kimberlin and Walker, 1986;
Kimberlin and Walker, 1989a; Kimberlin and Walker,
1989b; Lasmezas et al., 1996). Little is known about
the role of lymphocytes in the propagation of the
of
Fractionation
agent.
splenocytes from
CJD-infected mice revealed low-density lymphocytes
as preferential targets for agent replication. In vitro
stimulation of B and T cells with mitogens increased
susceptibility to CJD infection (Kuroda et al., 1983).
Similarly, mitogenic activation of the host enhanced
susceptibility to scrapie infection and conversely,
immunosuppressive treatment of mice reduced susceptibility to scrapie infection (Dickinson et al.,
1978). Within the framework of the "protein only"
hypothesis this could be explained by the fact that
mitogenic activation of lymphocytes results in
increased surface expression of PrP (Cashman et al.,
1990) thereby providing higher levels of substrate for
the conversion of PrPC into PrP Taken together, the
available data support a participation of cells of various lymphoid origins in agent propagation and disease pathogenesis.
sc.
LOCALIZATION OF PRION INFECTIVITY
IN THE LRS
To determine which cells in the LRS are carriers of
prions, we undertook to analyze the levels of prion
infectivity in various subsets of lymphoid cells isolated from scrapie-infected mice.
In a first experiment, we analyzed spleens of
wild-type (129/Sv-C57BL/6) mice 34 days after i.p.
inoculation with RML prions. At this time point,
prion levels reach a maximum in the spleen and stay
approximately constant during the whole incubation
period (Clarke and Haig, 1971). Spleens were fractionated into pulp and stroma. B and T cells, respectively, were purified from the pulp fraction by
magnetic-activated cell sorting (MACS), followed by
complement lysis of B cells in the T cell fraction and
vice versa. Finally, viable cells were isolated by density gradient centrifugation. This three-step procedure
led consistently to more than 99% pure T and B lymphocyte preparations, respectively, devoid of detectable cross-contamination, in 5-10% yield. In addition,
a non-B, non-T cell population was obtained by
depleting splenocytes of B and T lymphocytes by
PRION REPLICATION IN SPLEEN
Host
genotype
293
Donor
fetal liver cell
splenocytes
B cells
T cells
non B/T cells
Prnp+//
PBL
stroma
splenocytes
B cells
Prnp+/+
Prnpo/o
T cells
non B/T cells
PBL
stroma
2
3
5
log LD50 units/spleen
4
6
7
FIGURE Distribution of prion infectivity in spleen fractions and peripheral blood leukocytes of scrapie-infected mice. Serial 10-fold dilutions of splenocytes, splenocyte fractions, spleen stroma homogenate or PBLs were inoculated intracerebrally into groups of four indicator
mice and incubation time to terminal scrapie disease was determined. Infectivity titers were calculated from the data given in Raeber et al.
(1999a), assuming 3 108 lymphocytes/spleen, of which 65 % were B- and 35 % T-lymphocytes. The detection limit of the infectivity assay
corresponds to 100 LDs0 units per spleen
MACS; this fraction contained < 2% T but no detectable B lymphocytes (Raeber et al., 1999a). The cell
preparations and the stroma fraction were analyzed
for infectivity by endpoint titration (Brandner et al.,
1996; Fischer et al., 1996) (Figure 1). Total splenocytes had about 3.5 log LDs0 units per 106 cells and
both B and T cells showed infectivity titers within the
same order of magnitude, 3.4 and 3.5 log LDs0 units
per 106 cells, respectively. Strikingly, the non-B,
non-T cell population, which consists mainly of
monocytes and granulocytes (Binder et al., 1997)
contained only about 1 log LDs0 unit per 106 cells
(which could be attributed to the < 2% contamination
by T lymphocytes), arguing that prion infectivity in
the splenocyte fraction was not due to unspecific contamination with infectivity released from the stromal
fraction (Clarke and Kimberlin, 1984). The pulp of a
spleen contains 2-4 108 cells, about 80% of which
are lymphocytes (Binder et al., 1997). Inasmuch as
the purified B and T cells were representative of their
class as regards infectivity, about 300 x 3.5 log LDs0
units 6 log LDs0 units of infectivity were associated
with the pulp and about 4.3 log LD50 units with the
stroma of one spleen (Raeber et al., 1999a). Essentially all infectivity detected in total spleen extracts
was accounted for by the fractions. There was about
50 times more infectivity associated with lymphocytes than with stroma. Clarke and Kimberlin
(Clarke and Kimberlin, 1984) have reported about
equal distribution of infectivity between pulp and
stroma, however their data were obtained with different mouse and scrapie strains than ours.
Because these findings suggested that lymphocytes
might be responsible for spreading prions through the
organism, we determined the infectivity of peripheral
blood leukocytes (PBLs) from the same animals
whose spleens had been analyzed. We were surprised
that despite the relatively high infectivity associated
294
ALEX J. RAEBER et al.
with splenic B and T cells, intracerebral inoculation
of 106 PBLs from buffy coat did not cause disease in
any of 4 indicator mice (Raeber et al., 1999a).
Because about 80% of murine PBLs are lymphocytes
(Binder et al., 1997), less than 1 LDs0 unit of infectivity was associated with 8 105 lymphocytes; the specific infectivity of peripheral lymphocytes was
therefore at least 2-3000 times less that of splenic
lymphocytes. Because a mouse has about 2 107 circulating lymphocytes, there would be less than 20
LDs0 units of cell-associated infectivity, if any, circulating at one time.
Why is infectivity found on splenic but not on circulating lymphocytes? Perhaps only distinct subsets
of splenic lymphocytes carry infectivity and these do
not enter the blood stream or perhaps prion-carrying
splenic lymphocytes are eliminated in the spleen. As
regards the role of lymphocytes in the spread of infectivity from the periphery to the CNS, it would seem
that circulating, prion-bearing B cells are not
required, not only because they were not detected outside the LRS, but because reconstitution of irradiated
wild-type mice (Blittler et al., 1997) or immunodeficient mice (Klein et al,., 1998) with LRS devoid of
PrP restored invasion of the CNS by prions following
i.p. inoculation.
These findings demonstrate that splenic lymphocytes are carriers of prions. However, it is not
immediately clear whether prions associated with
splenic lymphocytes are synthesized de novo by these
cells or whether splenic lymphocytes are able to scavenge prions from other sources. These might include
other cells, which replicate or accumulate prions.
Unspecific contamination is unlikely because the specific infectivity of non-B, non-T cells derived from
the pulp was 2-3000 times less than that of B or T
lymphocytes.
PRP KNOCKOUT MICE OVEREXPRESSING
PRP IN THE LRS
To clarify the role of lymphocytes in prion replication
we targeted PrP expression specifically to lymphoid
cells in a mouse otherwise devoid of PrP and deter-
PrP
Expression
PrP promoter
phgPrP
pPrP-5’HG
Eu/IRF-1
enh/promoter
LRS
Ick promoter
Ig< promoter ORF
:
IgK enh
T cells
Ck
B cells
FIGURE
2 Schematic representation of half-genomic PrP transgenes driven by heterologous promoters. The genomic mouse Prnp
locus is shown on top. Construction of the "half-genomic" PrP vector (phgPrP) lacking the 12-kb intron 2 has been described. Promoter cassettes were inserted into the promoterless vector
pPrP-5’HG to yield plck-PrP-5’HG and pEt/IRF1-PrP-5’HG. The
construct for B cell-specific expression, plgK-PrP, contained a PrP
open reading frame (ORF) driven by the immunoglobulin K (IgK)
light chain promoter/enhancer
mined whether such transgenic mice could propagate
prions. Using appropriate expression vectors, we targeted PrP expression to three different cell types or
tissues in PrP null mice: the lymphoreticular system
in general (Raeber et al., 1999b), T lymphocytes
(Raeber et al., 1999b) or B lymphocytes (Montrasio
1999) (Figure 2).
To express PrP ectopically in the LRS we placed
the PrP coding sequence under the control of the
chain
IRF 1-promoter/immunoglobulin
heavy
enhancer (Et), which had been shown to lead to overexpression of a linked cDNA in the thymus, spleen
and bone marrow (Yamada et al., 1991). Two transgenic Prnp / mouse lines carrying this construct
were established and one of them, Tg94/IRF, was analysed for transgene expression. PrP expression in the
spleen of Tg94/IRF mice was more than 1000-fold
higher than in wild-type spleen and PrP in brain was
0.05 of that in wild-type (Raeber et al., 1999b). Thus,
PrP expression in the spleen of the transgenic mice
et al.,
was more than 20-fold higher than in brain, in marked
PRION REPLICATION IN SPLEEN
contrast to wild-type mice, where PrP levels in brain
exceed those in spleen about 100-fold. PrP on the surface of peripheral blood leukocytes, as determined by
cytofluorometry (FACS), was about 10-fold higher in
Tg94/IRF than in wild-type mice (Figure 3C). High
levels of PrP were also observed on B and T lymphocytes of Tg94/IRF splenocytes (Figure 3A) (Raeber et al., 1999b). Cryosections of spleen from
non-infected wild-type, Prnp / and Tg94/IRF mice
were doubly stained for germinal center B cells (with
peanut agglutinin, green) and PrP (with PrP antiserum
340, red). In wild-type spleens, PrP was mainly
present in germinal centers while in Tg94/IRF spleens
it was uniformly distributed over white and red pulp
(Figure 4 upper panel). In Figure 4 (lower panel)
consecutive spleen sections were labeled with the
FDC-specific antibody M1 (green) and PrP antiserum
(red; simultaneous staining did not succeed), again
revealing a striking overlap of FDC and PrP staining
within germinal centers in wild-type spleens. In
Tg94/IRF spleens, FDCs were stained in the germinal
centers whereas PrP-specific fluorescence was uniform over the whole section, which is compatible
with the FACS analys, is showing that B and T lymphocytes expressed PrP and with the assumption that
FDCs also expressed PrP (Raeber et al., 1999b).
Wild-type and Tg94/IRF mice hemizygous for the
transgene cluster were inoculated i.p. As shown in
Table I, two weeks after inoculation, spleen extracts
295
from Tg94/IRF mice and wild-type animals had the
same titer, about 7 log LD50 units/ml 10% homogenate and no infectivity was detected in brain. Six
months after inoculation the titers of Tg94/IRF spleen
extracts were essentially unchanged, somewhat
higher than the value of 6.5 for wild-type spleen and
no infectivity was detected in Tg94/IRF brains, as
compared to 8 log LD50 units/ml 10% homogenate
for wild-type. This demonstrates that following i.p.
challenge with mouse prions, Tg94/IRF mice maintained high titers of prions in the spleen from two
weeks up to six months after inoculation without
detectable infectivity appearing in the brain. It is
remarkable that despite the >1000 fold overexpression of PrP in the spleen of Tg94/IRF mice, the prion
titer in this organ was not higher than that in
wild-type mice. This apparent paradox can be
explained if the high PrP levels in Tg94/IRF spleen
reflected PrP in B and T lymphocytes but not in other
spleen cells such as FDCs and prion replication were
dependent on the latter. Alternatively, it is possible
that the level of PrP is not the limiting factor for the
formation of prions. Similarly, at the terminal stage of
scrapie, the prion titer in brains of tga20 mice, which
overexpress PrP 5-8 fold, is about the same as in
wild-type, although the incubation times for the transgenic and wild-type mice are 60 and 160 days, respectively (Fischer et al., 1996).
TABLE Scrapie infectivity in spleen and thymus of mice inoculated with high doses of RML prions
Genotype
PrP expression
Spleen titer
(log LD5o/m110% homogenate)1
2 weeks
Prnp
+/+
Prnp/
6 months
Thymus titer
(log LD5o/m110% homogenate) b
2 weeks
6 months
7
6.5
3.5
4.5
<1.5
<1.5
<1.5
<1.5
Tg94/IRF
LRS
7
7
nd
nd
Tg33/lck
T cells
1.5
<1.5
<1.5
<1.5
Tg306/lg:
B cells
<1.5
<1.5
nd
nd
All data are from Raeber et al. (1999b).
nd not done.
a.
b.
c.
Animals were inoculated intraperitoneally with
Bioassay limit of detection: 1.5 log LDs0/ml.
8 week time point.
106D50 of the RML strain of mouse prions.
ALEX J. RAEBER et al.
296
TABLE II Scrapie infeclivity in spleen and brain of mice following i.p. inoculation with low doses of RML prions
Spleen titer (log LD50/ml)
Genotype
Inoculum RML strain (log LD50
Prnp o/o
Prnp +/+
Prnp +/+
7
7b
3.5
3.5 1
Tg94/IRF
Bain ’titer (io’g LD5o/ml)a
2 weeks
8 weeks
12 weeks
12 weeks
<1.5
6.2
2.5
2
<1.5
6.9
6
5
<1.5
5.9
6
6
<1.5
6.8
nd
<1.5
nd not done.
Titers are expressed in log LDs0 units ml 10% homogenate.
Data from Biieler et al. (1993) Cell 73:1339.
c. Data from Clarke and Haig (1971) Res.vet.Sci. 12:195.
d. Data from Raeber et al. (1999) EMBO J. 18:2702.
a.
b.
e.
Measured at 6 months post inoculation.
PRIONS ARE REPLICATED IN THE LRS
Although high prion titers are found in spleen within
few days after i.c. or i.p. inoculation, it is not immediately clear whether this reflects de novo synthesis in the
LRS or scavenging of infectious agent generated in
brain or derived from the inoculum. Inoculation with
low prion doses had shown that net increase of infectious agent resulted in the spleen (Clarke and Haig,
1971), however it could, not be excluded that the agent
was being synthesized in the brain and transported to
the LRS. To resolve this question, we inoculated
Tg94/IRF mice i.p. with a low dose of RML prions (3.5
log LD50 i.c. units) and analyzed spleen homogenates
at various times after infection by endpoint titration. As
shown in Table II, prion titers in the spleen (in log
LD50 i.c. units/ml 10% homogenate) rose from 2 at two
weeks after inoculation to about 5 after 8 weeks and
remained at this level up to 12 weeks (Raeber et al.,
1999b). Because a spleen weighs about 100 mg, this
represents an increase of 4 logs, showing that prions
are replicated in the spleen of intraperitoneally inoculated Tg94/IRF mice and are not due to residual inoculum or import from the brain, which even at 6 months
contained no detectable infectivity.
ARE LYMPHOCYTES ABLE TO REPLICATE
PRIONS?
We have shown that the LRS efficiently replicates prions after i.p as well as after i.c. infection. To further
dissect the host cell types involved in prion replication in the LRS, we targeted PrP expression to lymphocytes and asked whether infectivity is synthesized
in the spleen and thymus of such transgenic mice.
PRP KNOCKOUT MICE OVEREXPRESSING
PRP IN T CELLS
Transgenic mouse lines with PrP expression restricted
to T lymphocytes were generated with the T-lymphocyte-specific Lck promoter (Chaffin et al., 1990).
PrP expression in Tg33/lck mice revealed PrP transcript levels in the thymus at least 50-fold higher than
in wild-type (data not shown). Significant levels of PrP
mRNA were also found in spleen and kidney. Tg33/lck
thymus and spleen had PrP levels that were at least
100-fold and 40-fold higher, respectively, than in
wild-type. PrP was undetectable in Tg33/lck brain
(Raeber et al., 1999b). The high level of PrP expression
on T lymphocytes was confirmed by FACS analysis of
Tg33/lck thymocytes (Figure 3B) and estimated to be
50-fold higher than in wild-type. No PrP expression
was detected in Tg33/lck splenic B lymphocytes
whereas splenic T lymphocytes were strongly positive
for PrP (Figure 3A). Immunohistochemical analysis of
Tg33/lck spleens (Figure 4, lower panel) showed that
PrP expression (red) was predominantly in the perifollicular T cell area while the germinal centers, where the
FDCs (green) were located showed little red fluorescence over background (Raeber et al., 1999b).
PRION REPLICATION IN SPLEEN
A
Prnp/
Tg94/IRF
297
Tg33/ick
thymocytes
B
C
fluorescence intensity
FIGURE 3 Analysis of PrP expression by FACS. Splenocytes (A), thymocytes (B) and peripheral blood leukocytes (PBL) (C) gated for lymphocytes from Prnp +/+ Prnp 0/0 Tg94/IRF and Tg33/lck mice. Cells were stained with anti-PrP polyclonal antisera R340 and phycoerythfin-conjugated anti-rabbit IgG and analyzed by FACS gated for lymphocytes. For two-colour FACS analysis (A), PrP staining was followed
by B cell staining with FITC-conjugated anti-B220 antibodies or T-cell staining with FITC-conjugated anti-CD3 antibodies. From Raeber et
al. (1999b)
To determine whether PrPC expression in T lymphocytes of Tg33/lck mice enabled prion replication
in thymus and spleen, we assayed tissue extracts
pooled from two animals sacrificed at 2 weeks, and 6
298
ALEX J. RAEBER et al.
PrP
{TR)
(F:TC>
<Tni
PNA (FtTG)
PP (TR)
iTn;
FIGURE 4 Analysis of PrP expression by immunohistochemistry. (UPPER PANEL) Double immunofluorescence analysis of splenic germinal centers in non-inoculated Tg94/IRF (a-d), wild-type mice (e-h), and Prnp/ mice (j-m). Sections were stained with haemalaun (a, e, j),
with peanut agglutinin (PNA) (green; b, f, k), and with antiserum R340 to PrP (red; c, g, 1). The majority of PNA-labeled germinal center
B-cells were PrP-positive in Tg94/IRF mice (d; yellow signal in superimposed images) and in wild-type mice (h), but PrP-negative in
Prnp/ mice (m). Original magnification 250x. (LOWER PANEL) Immunofluorescence labeling of follicular dendritic cells and PrP on
consecutive sections of spleen from non-inoculated Tg94/IRF (a-d), Tg33/lck (e-h), wild-type mice (j-m) and Prnp / mice (n-q). Sections
were stained with haemalaun (a, e, j, n), antibody FDC-M1 to follicular dendritic cell (green; b, f, k, o), antiserum R340 to PrP (red; c, g, 1, p)
and rabbit pre-immune serum (PIS) (d, h, m, q). In wild-type spleens (k, 1), PrP was stained exclusively in the germinal centers, most strongly
in the areas also stained by FDC-M1. In Tg94/IRF mice (b, c), PrP was evenly distributed over the entire section, including the region also
stained by FDC-M1. In Tg33/lck spleens, PrP was visualized mainly in the T cell areas but some cells were stained in the region also stained
by FDC-M1. No PrP staining above background (q) was found in germinal centers of Prnp/ mice (p). Original magnification 250x. From
Raeber et al. (1999b)
PRION REPLICATION IN SPLEEN
months, after i.p. inoculation. In the case of
scrapie-infected Tg33/lck mice, homogenates prepared from spleen two weeks after inoculation led to
disease in two out of four indicator CD-1 mice corresponding to a titer of about 1.5 log LDs0 units/ml
10% homogenate. Samples from thymus extracts produced disease in one out of four CD-1 mice giving a
titer of less than 1.5 log LDs0 units/ml 10% homogenate. No infectivity was detected in Tg33/lck spleen
or thymus 6 months after inoculation (Table I). Thymus homogenates from Prnp / mice also led to disease in one of four indicator mice. Most likely these
borderline infectivities are due to prions persisting
from the inoculum (Race and Chesebro, 1998; Sailer
et al., 1994). Two weeks and 6 months after i.p. inoculation thymus of wild-type mice had titers of about
3.5 and 4.5 log LDs0 units/ml 10% homogenate,
respectively (Raeber et al., 1999b). Thus even vast
overexpression of PrPc on T lymphocytes, comparable to levels found in wild-type brain, is not sufficient
to allow prion replication in thymus or spleen of
299
ined. The founder with the highest PrP expression
(Tg306/lg:) showed PrP levels on B cells corresponding to the level found on B cells of heterozygous Prnp /+ mice, about 50% of the wild-type.
Tg306/Ig: mice were inoculated i.p. with prions and
found to be completely resistant to scrapie (Montrasio
et al.,
1999).
To determine whether PrPC expression in B lymphocytes of Tg306/Igc mice enabled prion replication
in the spleen, we assayed spleen extracts from animals sacrificed at 2 and 8 weeks after i.p. inoculation.
No infectivity was detected in spleens from
scrapie-infected Tg306/Ig: mice (Table I) whereas
prion titers of around 106 LDs0 units per ml 10%
homogenate were found in spleen from
scrapie-infected Prnp /+ mice sacrificed at 2 and 8
weeks after inoculation (Montrasio et al., 1999).
Thus, B lymphocytes alone are not competent for
prion replication.
Prnp / mice.
PRP KNOCKOUT MICE OVEREXPRESSING
PRP IN B CELLS
It was previously shown that differentiated B cells are
crucial for neuroinvasion by prions and for replication
of the infectious agent in the LRS (Klein et al., 1997).
Whether B cells are directly involved in replicating
prions or rather B cell-dependent processes or factors
play a role remained uncertain. In order to determine
the capability of PrP-expressing B lymphocytes to
replicate prions, we generated transgenic mice with
PrP-expression restricted to B lymphocytes. The regulatory elements from an Ig kappa variable region
gene (Vk) were used to drive the transcription of a PrP
cDNA cloned with its own initiation codon and leader
sequence downstream of the V: promoter. Nine transgenic founders were established and it was shown by
FACS analysis that PrP expression was restricted to B
cells with no expression detectable on T cells. Northern blot analysis revealed a complete lack of PrP
expression in brain and several other tissues exam-
LYMPHOCYTES LACK A HOST
COMPONENT REQUIRED FOR PRION
REPLICATION
Using ectopic expression of PrP in B or T cells, we
showed that expression of PrPC on lymphocytes alone
is not sufficient to allow prion replication in thymus
or spleen of Prnp / mice. The following explanations
can be offered: (1) Lymphocytes are devoid of a conjectural receptor required for prion uptake or lack cellular factor(s) required for prion replication. Several
putative PrP receptors have been reported recently
(Martins et al., 1997; Rieger et al., 1997; Yehiely et
al., 1997), but have not been characterized at the
functional level. Experiments with transgenic mice
led to the suggestion that a species-specific factor X is
required for prion replication (Kaneko et al., 1997;
Telling et al., 1995). (2) Lymphocytes are able to replicate prions but are either rapidly eliminated due to a
prion-elicited toxic effect or "washed out" as a result
of normal turnover. (3) Finally, it is possible that prions administered i.p. in a PrP knockout environment
are not transported or transferred to lymphocytes.
300
ALEX J. RAEBER et al.
INVOLVEMENT OF STROMAL
COMPONENTS IN PRION REPLICATION
IN THE LRS
We have shown that prions are replicated in the LRS
but that lymphocytes seem to be unable to replicate
prions on their own. We have also found that lymphocytes isolated from spleens of scrapie-infected
wild-type mice are associated with prions (Raeber et
al., 1999a). Thus, it is possible that lymphocytes can
either acquire prions from a different cell type or replicate them in dependence of other cells. Because
Tg94/IRF mice, which express high levels of PrP on
lymphocytes, but also on other cells of the LRS, accumulate prions to high levels in spleen and thymus, PrP
expression on other splenocytes appears to be necessary for prion replication in the LRS.
To clarify the role of PrP expression on stromal
cells on prion replication in the LRS, we generated
mice whose bone marrow was chimeric with regard to
PrP expression. We lethally irradiated Prnp +/+ mice
and reconstituted them with fetal liver cells derived
from Prnp / mice. PCR analysis of splenocytes,
PBLs, stroma and tail tissue confirmed that these
mice had undergone successful reconstitution and
FACS analysis of lymphocytes demonstrated the
Prnp / origin of these cells. Spleens from these mice,
34 days after i.p. inoculation with RML prions, were
fractionated into pulp and stroma. B and T cells were
purified from the pulp fraction by magnetic-activated
cell sorting (MACS) followed by complement lysis of
B cells in the T cell fraction and vice versa as
described above. The cell preparations and the stroma
fraction were analyzed for infectivity by endpoint
titration. No infectivity was found in either total
splenocytes (<1 LDs0 unit per 106 cells), or in purified B or T lymphocytes (<1 LDs0 unit per 105 cells).
However, prion titers in the stromal fraction were
close to those in wild-type mice (Figure 1) (Raeber et
al., 1999a).
In the previous study aimed at localizing prion
infectivity in the LRS of wild-type mice, we found
prions associated with splenic lymphocytes. We then
asked whether association of infectivity with lymphocytes of scrapie-infected wild-type mice was spe-
cific or adventitious. Because lymphocytes
interdigitate with FDCs (Heinen et al., 1995), they
might have acquired prions or prion-containing,
tom-off membrane fragments from the latter during
the isolation procedure. However, the finding that
wild-type mice reconstituted with PrP-less FLCs
show no infectivity on splenic lymphocytes but significant prion titers in the stroma argues that if this
"transfer" hypothesis is correct, the postulated adhesion of scrapie agent is dependent on the presence of
PrP on lymphocytes; PrP would then function as
receptor for the infectious agent. But which cell-type
in the LRS replicates prions in the first place?
Prion infectivity in the stromal component of
peripheral lymphoid organs is thought to reside in
radiation-resistant post-mitotic cells (Fraser and Farquhar, 1987; Fraser et al., 1989). A prime candidate is
the follicular dendritic cell, because the normal isoform of the prion protein, PrPc seems to co-localize
with FDCs in uninfected mice (McBride et al., 1992)
while PrPsc co-localizes with FDCs in mice inoculated with CJD or scrapie agent (Kitamoto et al.,
1991; Klein et al., 1998). Thus, a likely source of prions in the LRS would be the FDCs (Brown et al.,
1999).
A MODEL FOR PRION REPLICATION
IN THE LRS
From studies of transgenic mice with ectopic PrP
expression and from the analysis of prion titers in
splenocytes of wild-type and bone marrow chimeric
mice, a picture of the mechanisms of prion replication
in the LRS is beginning to emerge (Figure 5). Following prion infection via peripheral routes, prions
are transported to and gain access to the LRS. It is
unclear which cells in the periphery support transport
of prions. Macrophages are possible candidates
because of their phagocytic activity and because their
mobility allows them to circulate between lymphoid
organs, blood and the parenchyma of many organs. In
lymphoid organs the earliest site of PrP sc accumulation and perhaps prion replication is the FDC.
PRION REPLICATION IN SPLEEN
301
Peripheral
Nemes
Prnp//+
Prnp+/+
PrnpO/O
PrPc
&:
Priori
(PrPso)
Priori replication
FIGURE 5 Model for prion replication in the LRS. Following prion infection of Prnp +// mice by the intraperitoneal route, prions are found
associated with B and T cells as well as with the stromal fraction containing FDCs. Prion replication in these mice likely occurs in FDCs or
other stromal cells or within the context of FDC and lymphocytes in the germinal center. Neuroinvasion of prions proceeds via infection of
nerve endings of the autonomic nervous system leading to infection of the peripheral and central nervous system where clinical disease
develops. In irradiated Prnp +// mice reconstituted with Prnp/ fetal liver cells (FLC), splenic lymphocytes fail to take up prions. But clinical disease in these mice develops with similar kinetics as in Prnp //+ mice suggesting that neuroinvasion of prions is not dependent on
prion-bearing lymphocytes but rather on the infection of peripheral autonomic nerve terminals
How prions accumulate and might replicate within
the context of the FDC network remains elusive.
Within the framework of the protein only hypothesis
prion replication could occur by conversion of PrPCexpressed on FDCs or neighboring lymphocytes
into further PrP sc molecules. No prion replication is
found in PrP knockout mice expressing PrP only on B
or T cells. Therefore, prions found associated with
PrP expressing splenic lymphocytes are either
acquired from FDCs (in a transfer dependent on PrP)
or else synthesized by the lymphocytes but only if
they are "infected" by prions presented by FDCs or
other stromal cells.
The mechanism by which PrP-expressing lymphocytes acquire prions remains unclear. Since lymphocytes are in close contact with FDCs in the
germinal center network it is likely that uptake of prions takes place through direct cellular interaction.
Horiuchi et al. (1999) have demonstrated binding of
PrPC to PrPSc in vitro, a step that precedes the conversion reaction. To summarize, a wealth of experimental
evidence supports an essential role of FDCs in prion
replication. Although it was shown that PrP sc (and
possibly prion infectivity) is associated with FDCs,
direct evidence for replication of prions in FDCs is
still lacking. All of the current model systems and
302
ALEX J. RAEBER et al.
assays to detect prions can not distinguish between
accumulation and replication of prions in FDCs.
Therefore, it would be important to develop a transgenic mouse model with ectopic PrP expression
restricted to FDCs. In addition the PrP transgene
could be engineered such that an epitope-tag in PrP
would allow to discriminate between de novo synthesized PrPsc and PrPsc derived from the inoculum.
Following prion replication in the LRS, prions
invade the CNS. Theoretically, there are two main
possibilities, haematogenous spread or neural spread.
The question whether lymphocytes play a role in the
spread of prions from the periphery to the CNS is of
more than academic interest. The extraordinary lymphotropism of vCJD prions (Hill et al., 1997) raises
new public-health concerns and demands urgently to
reassess the risk of transmitting vCJD through blood
or products thereof derived from individuals suffering
from pre-clinical prion disease. Our recent findings
suggest that at least in the mouse circulating
prion-bearing lymphocytes and in particular B cells
are not required to physically transport prions to the
brain. First of all, prion-bearing lymphocytes were
not detected in the blood of scrapie-infected mice
although splenic lymphocytes contained significant
levels of prion infectivity (Raeber et al., 1999a). More
importantly, reconstitution of irradiated wild-type
mice (Blittler et al., 1997) or immunodeficient mice
(Klein et al., 1998) with lymphohematopoietic stem
cells devoid of PrP restored invasion of the CNS by
prions following i.p. inoculation. Collectively, these
studies support the view that at least in the mouse
haematogenous spread of prions does not play a
major role in prion neuroinvasion. The essential role
of B lymphocytes in sustaining prion replication in
spleen and facilitating neuroinvasion may thus not be
that of replicating and/or transporting prions, but of
activating and maintaining activated FDCs (Fu et al.,
1998; Mackay and Browning, 1998; Matsumoto et
al., 1997).
Several lines of indirect evidence point to the
peripheral nervous system as the crucial compartment
that allows prions to get access to the central nervous
system (Kimberlin and Walker, 1979; Beekes et al.,
1996; Blittler et al., 1997). It is not clear whether pri-
ons can replicate in the peripheral nervous system or
whether they are simply transported along nerve fibers. Scrapie prions and PrPsc were found in the
peripheral nervous system of a scrapie-sick sheep
(Groschup et al., 1996) but so far PrP sc has not been
detected in the autonomic peripheral nervous system.
Interestingly, the innervation of lymphoid tissue is at
least in part controlled by lymphocytes themselves as
both T and B cells secrete nerve growth factor and on
the other hand nerve terminals secrete a variety of
molecules to stimulate the immune system (Straub et
al., 1998).
A thorough understanding of the role of the
immune system in peripheral prion pathogenesis is of
immediate importance in assessing the risk of iatrogenic transmission of prions and in the development
of diagnostic and therapeutic strategies for prion diseases.
Acknowledgements
This work was supported by the Kanton of Ztirich,
and by grants of the Scweizerischer Nationalfonds to
A.J.R., A.A. and C.W. and the European Union to
A.A. and C.W.
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