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From: TSS (216-119-143-170.ipset23.wt.net)
Subject: Scrapie transmission following exposure through the skin is dependent on follicular dendritic cells in lymphoid tissues [FULL TEXT]
Date: July 28, 2004 at 8:10 am PST
Scrapie transmission following exposure through
the skin is dependent on follicular dendritic cells
in lymphoid tissues [FULL TEXT] Joanne Mohan, Karen L. Brown, Christine F. Farquhar, Moira E. Bruce,
Neil A. Mabbott*
Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK
Received 9 March 2004; received in revised form 22 April 2004; accepted 12 May 2004
1. Introduction
The transmissible spongiform encephalophathies
(TSEs or prion diseases) are a group of infectious,
fatal, neurodegenerative diseases, which affect both
animals and humans. The precise nature of the TSE
agent is still subject to debate [1].However,PrPSc, an
abnormal, detergent-insoluble, relatively proteinase-
resistant isoform of a host glycoprotein PrPc
[2], is considered to constitute a major or possibly
the sole component of the infectious agent [3]. The
deposition of PrPSc within the brain of an infected
Journal of Dermatological Science (2004) 35, 101—111
KEYWORDS
Transmissible
spongiform
encephalopathy;
Scrapie;
Skin;
Follicular dendritic cell;
Prion protein;
Spleen
Summary Background: Transmissiblespongiformencephalopathies(TSEs)arechronic
infectious neurodegenerative diseases that are characterized by the accumulation in
affected tissues of PrPSc, an abnormal isoform of the host prion protein (PrPc). Following
peripheral exposure, PrPSc usually accumulates on follicular dendritic cells (FDCS) in
lymphoid tissues before neuroinvasion. Studies in mice have shown that TSE exposure
through scarified skin is an effectivemeans of transmission. Following inoculation via the
skin, a functional immune systemis critical for the transmission of scrapie to the brain as
severe combined immunodeficiency (SCID) mice are refractory to infection. Until now, it
was not knownwhich components of the immune systemare required for efficient scrapie
neuroinvasion following skin scarification. Objective: To determine which cells are
critical for the transmission of scrapie to the brain following inoculation via the skin.
Methods: A chimeric mouse model was used, which had a mismatch in PrPc expression
between FDCs and other bone marrow-derived cells within lymphoid tissues. These chimericmicewerechallengedwithscrapiebyskinscarificationtoallowtheseparaterolesof
FDCs and lymphocytes in peripheral scrapie pathogenesis to be determined. Results: We
show that mature FDCs are essential for the accumulation of scrapie within lymphoid
tissuesandthesubsequenttransmissionof infectiontothebrainfollowingTSEexposureby
this route. Furthermore, we show that the accumulation of PrPSc and infectivity in the
spleen is independent of PrP expression by lymphocytes or other bone marrow-derived
cells. Conclusion: Following inoculation with scrapie by skin scarification, replication in
the spleen and subsequent neuroinvasion is critically dependent upon mature FDCs.
2004 Japanese Society for InvestigativeDermatology.PublishedbyElsevier IrelandLtd.
All rights reserved.
*Corresponding author. Tel.: þ44 131 667 5204;
fax: þ44 131 668 3872.
E-mail address: neil.mabbott@bbsrc.ac.uk (N.A. Mabbott).
0923–1811/$30.00 2004 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jdermsci.2004.05.005
host correlates in most TSE diseases with the development
of neuropathological changes, such as vacuolation,
gliosis, and neuronal loss.
Many TSEs including natural sheep scrapie,
bovine spongiform encephalopathy (BSE), chronic
wasting disease in mule deer and elk, and variant
Creutzfeldt-Jakob disease (vCJD) in humans are
thought to be acquired by peripheral exposure.
For example, the consumption of BSE-contaminated
meat products by humans is most likely to be
responsible for the emergence of vCJD [4]. Following
peripheral inoculation with TSE agents, high
levels of infectivity and PrPSc usually accumulate
in lymphoid tissues prior to the dissemination of
infection to the central nervous system (CNS).
Within the lymphoid tissues of TSE-infected hosts
[5—10], PrPSc accumulation initially takes place in
germinal centres in association with follicular dendritic
cells (FDCs). Studies in rodents, inoculated
intra-peritoneally with scrapie, have shown that
mature FDCs are critical for scrapie accumulation
in lymphoid tissues. Furthermore, in the absence of
mature FDCs, the spread of disease to the CNS is
significantly impaired [7,8,11—14]. From the lymphoid
tissues, infectivity is translocated to the CNS
via peripheral nerves [15].
Although oral acquisition (ingestion) is considered
to be the main route of natural exposure to TSE
agents, other potential routes of exposure have
been identified. Sporadic sCJD in humans has been
transmitted iatrogenically through transplantation
of sCJD-contaminated tissues or pituitary-derived
hormones [16]. BSE and natural scrapie have also
been shown to be transmissible experimentally by
blood transfusion between sheep [17,18] raising
speculation that vCJD in humans might also be
transmitted via blood transfusion from an infected
donor. Studies in mice have shown that skin scarification
is an effective means of scrapie transmission
highlighting another possible route of exposure
[19]. For example, some cases of natural sheep
scrapie might be transmitted through sites of skin
scarification or skin lesions during close contact
with other scrapie-infected animals [20]. Scrapie
might also be established through skin or gum
lesions in the mouth [21], or be passed from mother
to offspring via the unhealed umbilical cord or
through sites of skin trauma at birth. Surgical instruments
contaminated with sCJD infectivity have also
been shown to have the potential to transmit disease
[22]. Animal models of TSE transmission
through scarified skin highlight important health
and safety issues, which include whether scientists
and health workers are at risk of acquiring infectivity
when handling infected tissues or instruments.
Biopharmaceutical and cosmetic products derived
from sheep and cattle tissues might harbour TSE
infectivity, with the potential to transmit disease
when applied to abraded skin [23,24]. Understanding
the immunobiology of scrapie transmission via
the skin will help in determining the possible significance
of this route in natural TSE infections and
aid the development of therapeutic strategies.
Previous studies have shown that a functional
immune system is critical for the translocation of
scrapie to the CNS following skin scarification, as
severe combined immunodeficiency (SCID) mice are
refractory to scrapie infection by this route [19].
Studies in this laboratory using the ME7 scrapie
strain have shown that following inoculation by
intra-peritoneal injection, mature FDCs are critical
for efficient neuroinvasion [7,8,13,44,45]. However,
whether FDCs or other components of the
immune system are required for efficient scrapie
neuroinvasion following inoculation by skin scarifi-
cation is not known. For example, as the skin is
highly innervated, it is plausible that lymphocytes
or Langerhans cells acquire scrapie within the skin
and transport it directly to peripheral nerves. To
maintain TSE infection, host cells must express the
cellular isomer of the host prion protein, as mice
deficient in PrPc (Prnp/ mice) do not develop
disease [25,26]. Thus, in order to determine which
cells are critical for the efficient transmission of
scrapie to the CNS following inoculation via the skin,
we used a chimeric mouse model previously established
in this laboratory, which had a mismatch in
PrPc status between FDCs and other bone marrowderived
cells within lymphoid tissues [7]. These
chimeric mice were challenged with scrapie by skin
scarification to allow the separate roles of FDCs and
lymphocytes in peripheral scrapie pathogenesis to
be determined.
2. Material and methods
2.1. Mice and bone marrow grafting
129/Ola mice were used as immunocompetent
wild-type controls. Bone marrow (BM) from the
femurs and tibias of immunocompetent adult
129/Ola (Prnpþ/þ) mice or PrP-deficient 129/Ola
(Prnp/) mice [26] was prepared as a single-cell
suspension (3 107 to 4 107 viable cells per ml) in
Hank’s balanced salt solution (Life Technologies,
Paisley, UK). Recipient SCID/Prnpþ/þ mice [7] were
reconstituted with 0.1 ml BM by injection into the
tail vein. Recipient mice and age-matched
ungrafted controls were used in subsequent experiments
28 days after BM grafting. All mice were
housed in individually ventilated cages to ensure
102 J. Mohan et al.
a high standard of microbiological hygiene. All protocols
using experimental rodents were approved by
the Institute’s Protocols and Ethics Committee and
carried out according to the strict regulations of the
UK Home Office ‘Animals (scientific procedures) Act
1986’.
2.2. Scrapie challenge
Mice were inoculated with the ME7 scrapie strain
by skin scarification of the medial surface of the left
thigh. Briefly, prior to scarification, approximately
1 cm2 area of hair covering the site of scarification
was trimmed using curved scissors and then
removed completely with an electric razor.
Twenty-four hours later a 23-gauge needle was used
to create a 5 mm long abrasion in the epidermal
layers of the skin at the scarification site. Then using
a 26-gauge needle, one droplet (6 ml) of ME7
scrapie inoculum from a 1% (w/v) terminal scrapie
mouse brain homogenate in physiological saline was
applied to the abrasion and worked into the site
using sweeping strokes. The scarification site was
then sealed with OpSite (Smith and Nephew Medical
Ltd., Hull, UK) and allowed to dry before the animals
returned to their final holding cages. Where
indicated separate groups of mice were inoculated
by intra-cerebral (i.c.) injection with 20 ml of the
same 1% scrapie mouse brain homogenate in physiological
saline (a dose of approximately 1 104:5
i.c. 50% infectious dose [ID50] units) as a titre control.
Following challenge, animals were coded,
assessed weekly for signs of clinical disease, and
killed at a standard clinical end-point [27]. Scrapie
diagnosis was confirmed by histopathological
assessment of TSE vacuolation in the brain. For
bioassay of scrapie infectivity, half spleens were
pooled from four animals from each group and
prepared as 10% (w/v) homogenates in physiological
saline and 20 ml injected i.c. into groups of 12 C57BL
indicator mice. The scrapie titer in each spleen was
determined from the mean incubation period in the
assay mice, by reference to established dose/incubation
period response curves for scrapie-infected
spleen tissue [28].
2.3. Immunohistochemical analysis
To monitor FDC status, spleen halves were snapfrozen
and stored at the temperature of liquid
nitrogen. Serial frozen sections (thickness, 10 mm)
were cut on a cryostat and FDCs were visualized by
staining with the FDC-specific rat monoclonal antiserum
FDC-M2 (AMS Biotechnology, Abingdon, UK),
or 8C12 monoclonal antiserum to detect CD35 (BD
PharMingen, Oxford, UK). B-lymphocytes were also
detected using the rat monoclonal antiserum B220
to detect CD45R (Caltag, Towcester, UK). Immunolabelling
was carried out using alkaline phosphatase
coupled to the avidin—biotin complex (Vector
Laboratories, Burlingame, CA, USA). Vector Red
(Vector Laboratories) was used as a substrate.
For the detection of PrP in brain tissue, tissues
were fixed in periodate-lysine-paraformaldehyde
and embedded in paraffin wax. Sections (thickness,
6 mm) were deparaffinized, and pretreated to
enhance PrP immunostaining by hydrated autoclaving
(15 min, 121 8C), and subsequent immersion in
formic acid (98%) for 5 min [5]. Sections were then
stained with the PrP-specific monoclonal antiserum
6H4 (Prionics, Zu¨rich, Switzerland) and immunolabelling
detected using hydrogen peroxidase coupled
to the avidin—biotin complex (Vector Laboratories)
with diaminobenzidine (DAB) as a substrate.
All sections were counterstained with hematoxylin
to distinguish cell nuclei.
2.4. Prnp genotype analysis of spleen tissue
Total DNA was extracted from spleen tissue fragments
(approximately 5 mg) by proteinase K digestion
and purified by phenol—chloroform extraction
by standard techniques. The Prnp genotypes of
spleen samples from BM-grafted animals were
determined by PCR analysis through the amplification
of the Prnp gene, and a portion of the neomycin
resistance gene to detect the presence of the
Prnp/ genotype [26]. The PCR mixture (total
volume 60.7 ml) contained; 5 ml of 10 PCR buffer,
5.0 ml of 50 mm MgCl2, 1.0 ml of 10 mm dNTP mix
(Life Technologies), 1 ml of chromosomal DNA,
0.5 ml (100 pmol/ml) of specific primers, 0.2 ml of
Taq polymerase (Life Technologies) and 50 ml of
sterile Dnase- and Rnase-free water. PCR analysis
was performed using the following specfic primers:
Prnp Fwd-50-ATG GCG AAC CTT GGC TAC TGG CTG-
30; Prnp Rev-50-TCA TCC CAG GAT CAG CAA GAT
GAG-30. These primers anneal to the start and stop
codons of the Prnp gene open reading frame,
respectively and generate a fragment of
750 bp. The sequences of the oligonucleotide primers
for the neomycin resistance gene were: Neo
Fwd-50-TTG AGC CTG GCG AAC AGT TC-30; Neo Rev-
50-GAT GGA TTG CAC GCA GGT TC-30. These primers
anneal to the neomycin resistance gene present in
Prnp/ mice, which is located within exon 3 of the
Prnp gene [26]. These primer pairs were designed to
generate a 550 bp fragment.
Following a hot start at 94 8C for 3 min, an
amplification cycle was carried out for 30 cycles
at the following temperatures: 94 8C for 50 s, 62 8C
for 50 s, 72 8C for 50 s on a thermal cycler (Genius
Scrapie transmission following exposure through the skin 103
PCR System, Techne, Cambridgeshire, UK). A final
extension period at 72 8C for 10 min was included at
the end of the 30 cycles. PCR products were
resolved by electrophoresis at 125 V through a
1.5% agarose gel containing 1 mg/ml ethidium bromide.
2.5. Immunoblot detection of PrPSc
Spleen fragments (approximately 20 mg) were
prepared as previously described by [29]. Briefly,
before immunoblot analysis, tissue homogenates
were treated in the presence or absence of 80 mg
proteinase K (to confirm the presence of PrPSc) and
subsequently partially purified by treatment with 2%
(w/v) N-lauroylsarcosine (in 0.1 M Tris—HCl pH 7.4),
allowing sedimentation only of the proteinase-Kresistant,
detergent-insoluble fraction of PrP
(PrPSc). Samples were subjected to electrophoresis
through sodium dodecyl sulphate 10—20% polyacrylamide
gels (Bio-Rad, Hemel Hempstead, UK) and
transferred to polyvinylidine difluoride membranes
(Bio-Rad) by semi-dry blotting. PrP was detected
with the PrP-specific mouse monoclonal antiserum
8H4 [30] (a kind gift from Professor Man-Sun Sy,
Case Western Reserve University School of Medicine,
Cleveland, OH, USA). Immunolabelling was
carried out using horseradish peroxidase-conjugated
rat anti-mouse antiserum (Jackson ImmunoResearch
Laboratories Inc., West Grove, PA, USA),
and bound horseradish peroxidase activity detected
with Supersignal West Dura Extended Duration
Substrate (Pierce, Chester, UK).
2.6. Statistical analysis
All data are presented as the mean S.E.M. and
error bars are indicated on figures where the S.E.M.
was 5% of the mean. The statistical significance of
differences in means of experimental groups was
calculated using ANOVA one-way analysis with Minitab
software. A P-value of 0.05 was considered to be
significantly different.
3. Results
3.1. Bone marrow reconstitution of
SCID/Prnpþ/þ mice restores scrapie
susceptibility following inoculation by
skin scarification
To test the hypothesis that mature FDCs are
critical for the translocation of scrapie to the CNS
following skin scarification, a chimeric mouse
model was used, which had a mismatch in PrP status
between its FDCs and lymphocyte populations [7].
The mouse models were produced by grafting SCID/
Prnpþ/þ mice with either PrP expressing (Prnpþ/þ)
or PrP-deficient (Prnp/—) BM from immunocompetent
129/Ola mice. As FDCs are not considered to be
derived from BM in adult mice [31,32], the lymphoid
tissues of SCID/Prnpþ/þ mice grafted with Prnp/
BM (SCID/Prnpþ/þ þ Prnp/ BM) will have PrP
expressing FDCs and other stromal-derived cells
but will lack PrP expression on lymphocytes. In
contrast, SCID/Prnpþ/þ mice grafted with Prnpþ/þ
BM (SCID/Prnpþ/þ þ Prnpþ/þ BM) will contain both
PrP expressing FDCs and lymphocytes.
Twenty-eight days after BM grafting, wild-type
129/Ola mice, SCID/Prnpþ/þ mice, SCID/Prnpþ/þ þ Prnpþ/þ BM mice and SCID/Prnpþ/þ þ Prnp/ BM
mice were inoculated with the ME7 scrapie strain by
skin scarification. All immunocompetent wild-type
129/Ola mice developed clinical signs of scrapie,
approximately 333 13 days post-inoculation
(n ¼ 12; Fig. 1). Characteristic disease-specific
PrP accumulation (Fig. 2a) and spongiform pathology
(Fig. 2e) typical of a peripheral infection with
the ME7 scrapie strain was detected in the brains of
all wild-type mice, which developed clinical disease.
In contrast, previous studies have shown that
SCID mice are refractory to challenge with ME7
scrapie by skin scarification up to 586 days postinoculation
[19]. Unfortunately, despite careful
husbandry, all ungrafted immunodeficient SCID/
Prnpþ/þ mice in this study succumbed to non-infectious,
non-TSE diseases (eg: thymic tumours) up to
274 days post-inoculation (Fig. 1). Immunohistochemical
analysis of brain tissue from all scrapieinoculated
SCID/Prnpþ/þ mice failed to detect any
signs of disease-specific PrP accumulation (Fig. 2b)
or vacuolation (Fig. 2f) consistent with the demonstration
that these mice are refractory to scrapie
following peripheral inoculation. However, the susceptibility
of most SCID/Prnpþ/þ mice to scrapie
infection was restored following grafting with
either Prnpþ/þ or Prnp/ BM (Fig. 1). Here, 6/11
SCID/Prnpþ/þ mice grafted with Prnpþ/þ BM developed
scrapie with a mean incubation period of 380 12 days post-inoculation. Likewise 8/10 SCID/
Prnpþ/þ mice grafted with Prnp/ BM developed
scrapie with a mean incubation period of 378 4
days post-inoculation. Characteristic spongiform
pathology and disease-specific PrP accumulation
were detected in the brains of all grafted SCID/
Prnpþ/þ mice, which succumbed to clinical disease
(Fig. 2).
No significant difference was observed between
the mean incubation periods of SCID/Prnpþ/þ mice
grafted with either Prnpþ/þ or Prnp/ BM. However,
a significant statistical difference was
104 J. Mohan et al.
observed between the mean incubation periods of
SCID/Prnpþ/þ mice grafted with either Prnpþ/þ BM
or Prnp/ BM when compared with wild-type mice
(P ¼ 0.014 and 0.004, respectively, ANOVA one-way
analysis). Grafted SCID/Prnpþ/þ mice developed
clinical scrapie approximately 47 days later than
the mean incubation period of immunocompetent
wild-type controls. However, no significant difference
in the pathological targeting of vacuolation in
the brain was observed between wild-type and
Fig. 2 Histological analysis of brain tissue from wild-type mice (a and e), SCID/Prnpþ/þ mice (b and f), SCID/Prnpþ/þ
mice reconstituted with Prnpþ/þ BM (SCID/Prnpþ/þ þ Prnpþ/þ BM; c and g), and SCID/Prnpþ/þ mice reconstituted with
Prnp/ BM (SCID/Prnpþ/þ þ Prnp/ BM; d and h) inoculated with scrapie by skin scarification. Large PrP
accumulations (brown; upper row) and spongiform pathology (H and E; lower row) were detected in the hippocampi of
all mice, which developed the clinical signs of scrapie. In contrast, no evidence of PrP accumulation (b) or spongiform
pathology (f) was detected in the brains of any SCID/Prnpþ/þ mice that succumbed to non-TSE diseases up to 274 dpi.
All sections were counterstained with hematoxylin (blue); pos.: mice that developed clinical signs of scrapie; neg.:
mice that were free of the signs of scrapie. Original magnification 200.
Fig. 1 Reconstitution of SCID/Prnpþ/þ mice with immunocompetent BM restores susceptibility to scrapie when
inoculated by skin scarification. Wild-type (WT) mice ( ); SCID/Prnpþ/þ mice, SCID/Prnpþ/þ mice reconstituted with
Prnpþ/þ BM ( ; SCID/Prnpþ/þ þ Prnpþ/þ BM) and SCID/Prnpþ/þ mice reconstituted with Prnp/ BM (&; SCID/Prnpþ/þ
þ Prnp/ BM) were inoculated with the ME7 scrapie strain by skin scarification (scarif.). Wild-type mice were also
inoculated by i.c. injection as a titre control ( ; WT i.c). Each bar represents the mean incubation period S.E.M. (*)
Incubation periods for individual mice that succumbed to clinical scrapie. (*) Times at which SCID/Prnpþ/þ mice
succumbed to non-TSE related disease.
Scrapie transmission following exposure through the skin 105
grafted SCID/Prnpþ/þ mice suggesting that neuroinvasion
had occurred via a common pathway in each
case.
Although the susceptibility of most SCID/Prnpþ/þ
mice to scrapie was restored following BM grafting,
5/11 SCID/Prnpþ/þ þ Prnpþ/þ BM mice and 2/10
SCID/Prnpþ/þ þ Prnp/ BM mice remained free
from the signs of scrapie 521 days post-inoculation,
at which point the experiment was terminated
(Fig. 1). Successful reconstitution and normal germinal
centre architecture were confirmed in these
mice (data not shown); however, immunohistochemical
analysis of brain tissue from all surviving
mice failed to detect any spongiform change or
disease-specific PrP accumulation (data not shown).
Likewise, no PrPSc accumulation was detected in
the spleen by immunoblot analysis (data not shown)
suggesting these mice would not have developed
clinical scrapie at a later stage.
3.2. Confirmation of immune status and
germinal centre architecture
Spleens and serum were taken from all mice to
monitor immune status. Consistent with the
absence of B-lymphocytes in SCID mice [33,34],
serum from all ungrafted SCID/Prnpþ/þ mice contained
barely detectable levels of immunoglobulin
(Ig) when compared with those of wild-type mice
(data not shown). However, ELISA analysis con-
firmed that reconstitution of SCID/Prnpþ/þ mice
with immunocompetent BM from either Prnpþ/þ
or Prnp/ mice restored serum Ig levels to those
observed in wild-type mice (data not shown). Thus
functional BM-derived B-lymphocytes had been successfully
grafted into recipient SCID/Prnpþ/þ mice.
We next determined the Prnp genotype in the
spleens of grafted mice by PCR analysis of total
splenic DNA (Fig. 3). Analysis of DNA from SCID/
Prnpþ/þ mice grafted with Prnpþ/þ BM detected
the presence of only the Prnp gene by the visualisation
of a single band at 750 bp (Fig. 3, lanes 1—4). In
contrast, two bands were detected in splenic DNA
samples from SCID/Prnpþ/þ mice grafted with
Prnp/ BM, demonstrating the presence of both
the Prnp gene (750 bp) and the neomycin resistance
gene (550 bp) (Fig. 3, lanes 5—8). Thus, these results
confirmed the presence of only Prnpþ/þ cells within
the spleens of SCID/Prnpþ/þþPrnpþ/þ BM mice, and
thepresenceofbothPrnpþ/þandPrnp/cellswithin
the spleens of SCID/Prnpþ/þ þ Prnp/ BM mice.
The germinal centre architecture in the spleen
was analysed by immunohistochemistry. As
expected, FDC-M2 and CD35 expressing FDC networks
and B-lymphocytes (CD45R/B220) were
detected in the spleens of all immunocompetent
wild-type mice (Fig. 4). B-lymphocytes produce
important factors for the maintenance and maturation
of FDCs. In the absence of B-lymphocytes FDCs
do not receive these important stimuli and do not
mature [33,34]. Thus mice deficient in B-lymphocytes
are indirectly deficient in FDCs. As Fig. 4
illustrates, both FDC networks and B-lymphocytes
were absent in spleens of ungrafted SCID/Prnpþ/þ
mice, consistent with the immunodeficent phenotype
of SCID mice [33,34]. However, FDC networks
in the spleens of SCID mice can be restored following
grafting with B-lymphocytes or immunocompetent
BM as a source of lymphocytes [34]. Likewise,
mature FDC networks and B-lymphocytes were
restored in spleens of SCID/Prnpþ/þ mice following
grafting with either Prnpþ/þ or Prnp/ BM (Fig. 4).
Thus, the restoration of germinal center architecture
in the lymphoid tissues of SCID/Prnpþ/þ mice,
following BM grafting, coincided with the restored
susceptibility of these mice to scrapie.
Fig. 3 Confirmation of the Prnp genotype in the spleens of SCID/Prnpþ/þ mice reconstituted with either Prnpþ/þ BM
(lanes 1—4) or Prnp/ BM (lanes 5—8). Analysis of total splenic DNA from SCID/Prnpþ/þ mice reconstituted with Prnpþ/þ
BMconfirmed the presence only of the Prnp gene by the visualisation of a single band at 750 bp (lanes 1—4). The visualisation
of two bands at 750 and 550 bp (lanes 5—8) confirmed the presence of both Prnp gene and a portion of the neomycin
resistance gene (Neomycin), respectively,within splenic DNAsamples fromSCID/Prnpþ/þ mice reconstituted with Prnp/
BM (SCID/Prnpþ/þ þ Prnp/ BM). Lane M, 200 bp molecular size markers. Controls included: C1, splenic DNA froma SCID/
Prnpþ/þ þ Prnp/ BM mouse; C2, splenic DNA from a wild-type/Prnpþ/þ mouse; W, PCR-amplified water was used as a
negative control.
106 J. Mohan et al.
3.3. Scrapie infectivity and PrPSc
accumulation in the spleen
Following peripheral inoculation with the ME7
scrapie strain, high levels of infectivity and the
disease-specific isoform of the prion protein, PrPSc,
accumulate in the spleen within 42 days post-inoculation
and are maintained throughout the course of
infection [29]. Spleen samples were taken from four
mice from each experimental group of mice 220
days post-inoculation with scrapie via skin scarification.
The scrapie infectivity titre in spleen lysates
from each group was estimated by bioassay in
groups of 12 indicator mice. As expected, spleens
from scrapie-inoculated wild-type mice contained
high levels of infectivity (approximately 5.7 log i.c.
ID50/g). In contrast, scrapie infectivity was undetectable
in spleen samples from ungrafted SCID/
Prnpþ/þ mice assayed 220 days post-inoculation
suggesting a scrapie infectivity titre, if present,
below 2.6 log i.c. ID50/g (at least 1000-fold less
than the level detected in spleens of wild-type mice
assayed at the same time post-inoculation). Scrapie
infectivity accumulation was restored in the spleens
of SCID/Prnpþ/þ mice following grafting with either
Prnpþ/þ or Prnp/ BM to the same magnitude
observed in wild type mice at the same time point
(approximately 6.7 and 5.7 log i.c. ID50/g, for
SCID/Prnpþ/þ mice grafted with either Prnpþ/þ or
Prnp/ BM, respectively).
Similarly, immunoblot analysis of spleen tissue
from terminally affected wild-type mice detected
large accumulations of detergent-insoluble proteinase-
K-resistant PrPSc (Fig. 5). A typical threebanded
pattern was observed between molecular
mass values of 20—30 kDa, representing the unglycosylated,
monoglycosylated, and diglycosylated
isomers of PrP (in order of increasing molecular
mass). However, no PrPSc accumulation was detectable
within the spleens of any ungrafted SCID/
Prnpþ/þ mice assayed at various times after inoculation
(Fig. 5a, lanes 4, 6 and 8). In comparison,
PrPSc accumulation was restored in the spleens of
terminally scrapie-affected SCID/Prnpþ/þ mice
grafted with either Prnpþ/þ or Prnp/ BM to levels
similar to those observed in wild type mice (Fig. 5b).
Fig. 4 Immunohistochemical analysis of the germinal centre architecture in spleen tissue from wild-type mice, SCID/
Prnpþ/þ mice, SCID/Prnpþ/þ mice reconstituted with Prnpþ/þ bone marrow (SCID/Prnpþ/þ þ Prnpþ/þ BM), and SCID/
Prnpþ/þ mice reconstituted with Prnp/ bone marrow (SCID/Prnpþ/þþ Prnp/ BM). Adjacent frozen sections were
stained with FDC-M2 antiserum to detect FDCs (red; a—d), CD35-specific monoclonal antiserum 8C12 to detect
complement receptor 1 (red; e—h) and the CD45R-specific antiserum B220 to detect B-lymphocytes (red; i—l). All
sections were counterstained with hematoxylin (blue). As expected, FDC-M2 and CD35 expressing FDC networks and Blymphocytes
(CD45R/B220) were detected in the spleens of all immunocompetent wild-type mice (a, e and i,
respectively). In the absence of B-lymphocytes, FDCs do not receive important stimuli and can not mature. Thus mice
deficient in B-lymphocytes are indirectly deficient in FDCs. As panels b, f, and j illustrate, both FDC networks and
B-lymphocytes were absent in spleens of ungrafted SCID/Prnpþ/þ mice. However, mature FDC networks and
B-lymphocytes were restored in spleens of SCID/Prnpþ/þ mice following grafting with either Prnpþ/þ (c, g, and k) or
Prnp/ BM (d, h, and l). Original magnification 400 (a—h), 200 (i—l).
Scrapie transmission following exposure through the skin 107
4. Discussion
Previous studies have shown that skin scarification
is an effective means of scrapie transmission in
immunocompetent mice. However, immunodefi-
cient SCID mice are refractory to scrapie when
inoculated by this route, illustrating that a functional
immune system is critical for the transmission
of scrapie to the CNS following inoculation via the
skin [19]. In this study, we have demonstrated that
reconstitution of SCID mice with immunocompetent
BM restores scrapie replication within lymphoid
tissues following skin scarification. This effect coincided
with the induction of FDC network maturation
within the spleens of grafted SCID mice and subsequent
ability to accumulate high levels of scrapie
infectivity and PrPSc. Furthermore, we have shown
that following inoculation via the skin, scrapie accumulation
in lymphoid tissues and subsequent translocation
to the CNS is dependent on mature FDCs
but independent of the PrP status of lymphoytes and
other BM-derived cells. Taken together, these findings
are consistent with previous studies, which
demonstrate that following intra-peritoneal inoculation
with the ME7 scrapie strain, a functional
immune system and more critically PrP-expressing
FDCs, are required for transport of the agent from
the periphery to the CNS [7,8,13].
SCID mice suffer from a congenital syndrome,
which is characterised by the loss of both B- and Tlymphocyte
immunity [33]. Secondary to this
defect, they also lack functional FDCs as stimulation
from lymphocytes is required for the maturation
and maintenance of FDCs [34]. Unfortunately,
despite careful husbandry due to their dysfunctional
immune system, all scrapie-challenged SCID/Prnpþ/
þ mice in this study succumbed to non-TSE related
disease up to 274 days post-inoculation. These diseases
were non-infectious (e.g. thymic tumours)
and were not a reflection of the microbiological
status of the husbandry conditions, which were
maintained to a high standard of hygiene. Previous
data from this laboratory [19] have shown that in
contrast to wild-type mice, SCID mice did not succumb
to clinical scrapie following exposure to a
similar dose of scrapie via skin scarification (mean
survival period ¼ 442 21 days post-inoculation,
n ¼ 23, range ¼ 259—586 days). Following intraperitoneal
inoculation with ME7, scrapie PrPSc is
detected in the brain considerably before the onset
of clinical signs [29]. In the current study, we
measured the levels of disease-specific PrP accumulations
in the spleens and brains of all scrapieinoculated
SCID mice. No disease-specific PrP accumulation
was detected within the brains or spleens
of any of the scrapie-inoculated SCID mice, supporting
the assumption that they would not have subsequently
developed clinical disease consistent
with data from previous studies using this TSE strain
[19]. Scrapie infectivity was also undetectable in
the spleens of scrapie-inoculated SCID mice,
Fig. 5 Immunoblot analysis of spleen tissue from
terminally scrapie-affected wild-type mice (WT), SCID/
Prnpþ/þ mice, SCID/Prnpþ/þ mice reconstituted with
Prnpþ/þ bone marrow (SCID/Prnpþ/þ þ Prnpþ/þ BM),
and SCID/Prnpþ/þ mice reconstituted with Prnp/ bone
marrow (SCID/Prnpþ/þ þ Prnp/ BM). Treatment of
tissue in the absence () or presence (þ) of proteinase K
(PK) prior to electrophoresis is indicated. After PK
treatment, a typical three-band pattern was observed
between molecular mass values of 20 and 30 kDa,
representing unglycosylated,monoglycosylated, and diglycosylated
isomers of PrP (in order of increasing molecular
mass). PrP was detected using the PrP-specific monoclonal
antiserum 8H4. (a) High levels of PrPSc were detected in
the spleens of terminally scrapie-affected WT mice, but
none was detected in tissues from SCID/Prnpþ/þ mice at
any time point. (b) However, high levels of PrPSc were
detected in spleens of SCID/Prnpþ/þ mice grafted with
either Prnpþ/þ (SCID/Prnpþ/þ þ Prnpþ/þ BM) or Prnp/
BM (SCID/Prnpþ/þ þ Prnp/ BM). Lane B is blank; pos.:
mice that developed clinical signs of scrapie; neg.: mice
that were free of the signs of scrapie; dpi: day postinoculation
on which the tissues were analysed.
108 J. Mohan et al.
assayed 220 days post-inoculation. These data are
consistent with the hypothesis that following inoculation
by skin scarification, scrapie infectivity is
unlikely to reach the CNS by direct transport via
nerves within the skin or via the bloodstream.
Engraftment of SCID/Prnpþ/þ mice with immunocompetent
BM-restored functional lymphocyte
populations within the spleen. Furthermore, these
lymphocytes were functional as they produced
immunoglobulins and were able to stimulate FDC
maturation and network formation [33,34]. The
development of germinal centre architecture, comparable
to immunocompetent animals, coincided
with restored scrapie susceptibility and the accumulation
of infectivity and PrPSc in lymphoid tissues
of these mice. Thus, these data demonstrate that
following inoculation through scarified skin, scrapie
accumulates in lymphoid tissues prior to neuroinvasion,
as observed with other peripheral routes of
exposure [7,9,35,36]. Our studies also demonstrate
that PrPc expression on FDCs alone in lymphoid
tissues is sufficient to establish scrapie infection.
In the presence of PrPc-expressing FDCs, the PrPc
status of bone marrow-derived cells had no signifi-
cant effect on the accumulation of infectivity and
PrPSc in the spleen or on disease incubation period.
Further experiments are necessary to determine
whether following inoculation via the skin, PrPcexpressing
lymphocytes are permissive to scrapie
replication in the absence of PrPc expression by
FDCs. However, these results are consistent with
the demonstration that PrPc expression on FDCs, not
lymphocytes, is critical for the peripheral accumulation
and transport of scrapie [7,12,37]. Thus, we
consider that lymphocytes would likewise be unlikely
to play a key role following inoculation via the
skin. Although not directly involved in the replication
of ME7 scrapie strain, lymphocytes play an
important indirect role in pathogenesis by maintaining
the maturation of FDC networks within lymphoid
tissues [33,34].
Interestingly, grafted SCID/Prnpþ/þ mice did display
significantly longer incubation periods in comparison
to immunocompetent wild-type mice.
Similar results have also been observed in grafted
SCID mice inoculated with scrapie strain C506M3 by
intra-peritoneal injection [38]. The reason for the
delay in the onset of the neurological disease in BMgrafted
SCID/Prnpþ/þ mice is not known, but it
might be that at the time of scrapie inoculation
the restoration of geminal centre functionality in
these mice was incomplete. Incomplete reconstitution
of SCID/Prnpþ/þ mice might be a consequence
of the high natural killer cell activity within SCID
mice, which may impair the development of donor
BM cells [39]. This effect could have reduced the
number of potential peripheral target cells, such as
FDCs, available for agent replication at the time of
inoculation. In the temporary absence of FDCs at
the time of inoculation, both scrapie replication in
the spleen and subsequent neuroinvasion are significantly
delayed [13]. To achieve efficient reconstitution,
it has been suggested that mice are sublethally
g-irradiated prior to cell transfer, to encourage
full maturation of grafted bone marrow cells
[40]. This procedure was not undertaken in this
study as SCID mice have an increased sensitivity
to irradiation due a general defect in DNA repair
mechanisms, which is believed to be closely linked
to the scid mutation [41]. It was our concern that girradiation
may have adverse affects on the architecture
of the skin, blood-brain barrier, or nervebrain
barrier of SCID mice; any of which may have
facilitated neuroinvasion by an atypicalmechanism.
Although neuroinvasion was delayed in the reconstituted
SCID/Prnpþ/þ mice, no significant difference
was observed in the severity or distribution of
vacuolation or disease-specific PrP accumulation
within the brains of wild-type and grafted SCID
mice, suggesting that neuroinvasion had occurred
in these mice via a common pathway.
Although the grafting of SCID/Prnpþ/þ mice with
immunocompetent BM-restored scrapie susceptibility
in most cases, 5/11 SCID/Prnpþ/þ þ Prnpþ/þ BM
mice and 2/10 SCID/Prnpþ/þ þ Prnp/ BM mice
remained free from the signs of scrapie 521 days
post-inoculation, at which point the experiment
was terminated. ELISA and PCR genotyping analysis
suggested that reconstitution had been successful in
these surviving mice (data not shown), yet they
were refractory to peripheral inoculation. Studies
suggest that following BM grafting of SCID mice, it
takes approximately 4—6 weeks for full restoration
of BM-derived cell populations and germinal centre
architecture [40]. As this time period may vary
between individual animals, it is conceivable that
in this study, the surviving grafted SCID/Prnpþ/þ
mice may not have achieved a mature, fully functional
immune status, prior to inoculation. This may
have prevented scrapie replication establishing due
to a lack of functional peripheral target cells such as
FDCs [13]. In the absence of mature FDCs at the time
of inoculation, it is likely that a significant amount
of the inoculum is destroyed by macrophages
[42,43]. This delay is again consistent with the
hypothesis that scrapie infectivity is unlikely to
reach the CNS from the skin by the direct capture
of infectivity by nerves within the skin or by direct
transport via the bloodstream.
How scrapie is transported from the skin to FDCs
within draining lymphoid tissues is not known.
Migratory BM-derived Langerhans cells are a plau-
Scrapie transmission following exposure through the skin 109
sible candidate mechanism as they acquire antigens
in the skin and transport them to lymphoid tissues.
Data presented here demonstrate that following
inoculation via the skin, the accumulation of scrapie
in the spleen and disease incubation period are not
affected by the PrPc status of bone marrow-derived
cells. Thus, if scrapie is transported from the skin to
lymphoid tissues in a cell-dependent manner, these
data suggest that PrPcexpression by such cells is not
critical.
Data presented here demonstrate that scrapie
replication in the spleen following inoculation by
skin scarification occurs only in the presence of
mature FDCs and is independent of the PrP status
of surrounding splenic lymphocytes and other bone
marrow-derived cells. Furthermore, our results
indicate that mature, functional FDCs are required
for subsequent neuroinvasion. These data are consistent
with previous research using the ME7 scrapie
strain, which suggests that FDCs are critical for
efficient scrapie neuroinvasion following intra-peritoneal
exposure [7,8,13]. Once TSEs spread to the
CNS, the neurodegeneration they cause is considered
irreversible. The identification of an important
role for FDCs in the pathogenesis of disease following
skin scarification provides an opportunity for
therapeutic intervention prior to neuroinvasion as
are already being investigated following other peripheral
routes of exposure [13,14,44,45].
Acknowledgements
We thank Jenny Beaton, Lorraine Gray, Irene
McConnell and Mary Brady (Institute for Animal
Health, Neuropathogenesis Unit, Edinburgh, UK)
for excellent technical support; Man-Sun Sy (Case
Western Reserve University School of Medicine,
Cleveland, OH, USA) for provision of 8H4 monoclonal
antiserum. This work was supported by funding
from the Medical Research Council and the Biotechnology
and Biological Sciences Research Council.
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