Báo cáo khoa học: CpG and LPS can interfere negatively with prion clearance in macrophage and microglial cells pdf

CpG and LPS can interfere negatively with prion clearance
in macrophage and microglial cells
Sabine Gilch
1
, Frank Schmitz
2
, Yasmine Aguib
1
, Claudia Kehler
1
, Sigrid Bu
¨
low
1
, Stefan Bauer
3
,
Elisabeth Kremmer
4
and Hermann M. Scha
¨
tzl
1
1 Institute of Virology, Prion Research Group, Technical University of Munich, Germany
2 Institute of Microbiology and Immunology, Technical University of Munich, Germany
3 Institute of Immunology, Philipps-University Marburg, Germany
4 GSF-National Research Centre for Environment and Health, Institute of Molecular Immunology, Munich, Germany
Prion diseases are fatal neurodegenerative disorders,
including scrapie in sheep, bovine spongiform encepha-
lopathy in cattle and Creutzfeldt–Jakob disease in
humans. They are characterized by the accumulation

of an abnormally folded isoform of the cellular prion
protein PrP
c
, designated PrP
Sc
, which appears to be
the causative agent of disease [1–4]. PrP
c
is a glycopro-
tein expressed rather ubiquitously, with the highest
expression levels found in the central nervous system.
It is linked to the outer leaflet of the plasma membrane
by a glycosyl-phosphatidyl-inositol anchor (reviewed in
[5]). Expression of PrP
c
is crucial for the development
of prion diseases, as mice ablated for the prnp gene do
not succumb to the disease [6]. The structure of soluble
PrP
c
is mainly a-helical [7]. During prion conversion, it
interacts with PrP
Sc
molecules and is re-folded to a
protein with a high b-sheet content, prone to aggrega-
tion [8,9]. This probably occurs at the plasma mem-
brane or in the early endocytic pathway, but the exact
subcellular site of prion conversion has not been iden-
tified [10–12].
The infectious agent in prion diseases seems to

consist solely of protein, underlined recently by studies
showing that prion infectivity can be generated in vitro
Keywords
innate immunity; prion; prion clearance;
PAMP; toll-like receptor
Correspondence
H. M. Scha
¨
tzl, Institute of Virology, Prion
Research Group, Technical University of
Munich, Trogerstr. 30, 81675 Munich,
Germany
Fax: +49 89 41406823
Tel: +49 89 41406820
E-mail:
(Received 1 July 2007, revised 9 September
2007, accepted 13 September 2007)
doi:10.1111/j.1742-4658.2007.06105.x
Cells of the innate immune system play important roles in the progression
of prion disease after peripheral infection. It has been found in vivo and
in vitro that the expression of the cellular prion protein (PrP
c
) is up-regu-
lated on stimulation of immune cells, also indicating the functional impor-
tance of PrP
c
in the immune system. The aim of our study was to
investigate the impact of cytosine-phosphate-guanosine- and lipopolysac-
charide-induced PrP
c

up-regulation on the uptake and processing of the
pathological prion protein (PrP
Sc
) in phagocytic innate immune cells. For
this purpose, we challenged the macrophage cell line J774, the microglial
cell line BV-2 and primary bone marrow-derived macrophages in a resting
or stimulated state with various prion strains, and monitored the uptake
and clearance of PrP
Sc
. Interestingly, stimulation led either to a transient
increase in the level of PrP
Sc
relative to unstimulated cells or to a deceler-
ated degradation of PrP
Sc
. These features were dependent on cell type and
prion strain. Our data indicate that the stimulation of innate immune cells
may be able to support transient prion propagation, possibly explained by
an increased PrP
c
cell surface expression in stimulated cells. We suggest
that stimulation of innate immune cells can lead to an imbalance between
the propagation and degradation of PrP
Sc
.
Abbreviations
BMDM, bone marrow-derived macrophage; CpG, cytosine-phosphate-guanosine; FACS, fluorescence-associated cell sorting; FDC, follicular
dendritic cell; FITC, fluorescein isothiocyanate; LPS, lipopolysaccharide; ODN, oligodeoxynucleotide; PAMP, pathogen-associated molecular
pattern; PK, proteinase K; PrP
c

, cellular prion protein; PrP
Sc
, abnormally folded isoform of the cellular prion protein; RML, Rocky Mountain
Laboratory strain of mouse-adapted scrapie; TLR, Toll-like receptor; TNF-a, tumour necrosis factor-a.
5834 FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS
[13,14]. On peripheral infection, a huge body of evi-
dence points to the role of immune cells in the neuro-
invasion process [15–18]. Transport through epithelial
colon cells in the presence of differentiated M-cells
may enable prions to gain first access to the lympho-
reticular system [19]. Furthermore, migrating intestinal
dendritic cells, B-cells and resident follicular dendritic
cells (FDCs) play a role in the development of prion
disease after peripheral infection [20–22], with FDCs
being the cells in which prion propagation occurs in
the spleen. It has been shown that various dendritic
cell subsets can degrade PrP
Sc
[23–25] and, also, can
transport PrP
Sc
, but their importance for neuroinva-
sion is still controversial [15,22,25–27]. By contrast,
macrophages may be involved in the clearance of pri-
ons [28–31]. Microglial cells are resident brain macro-
phages and become activated during the progression of
prion disease [32]. They contribute to the neurodegen-
erative phenotype of prion diseases by producing
inflammatory cytokines in mouse models, although the
response is apparently dependent on the prion strain

used for infection [33,34]. Microglial cells can contain
infectivity in vivo and may disseminate prion infectivity
within the brain during their migratory activities [35].
Recently, a microglial cell line derived from PrP over-
expressing mice has been established which can be
infected with several prion strains [36].
The physiological role of PrP
c
has not yet been
clarified. Some evidence indicates a functional role of
PrP
c
in the immune system. The expression of PrP
c
is up-regulated, e.g. on maturation of nonplasmacy-
toid dendritic cells, on activated T-cells or on inter-
feron-c-treated monocytes [37–40]. Immunization of
mice with vesicular stomatitis virus led to an up-regu-
lation of PrP
c
in the FDC network [41]. When
attached to the surface of monocyte ⁄ macrophage
cells, fusion proteins of the prion protein activated
downstream signalling [42]. Macrophages derived
from prnp knock-out mice exhibited a decreased
phagocytic activity in vitro [43].
In this study, we sought to investigate the impact of
stimulation-induced PrP
c
up-regulation in macrophage

or microglial cell lines and primary macrophages on the
processing of PrP
Sc
. We used the macrophage cell line
J774, the microglial cell line BV-2 and mouse bone mar-
row-derived macrophages (BMDMs). On activation
with cytosine-phosphate-guanosine-oligodeoxynucleo-
tides (CpG-ODNs) or lipopolysaccharide (LPS), cells
showed an up-regulation of PrP
c
of about twofold with
similar kinetics. There were distinct differences in the
reaction to prion infection, but, in all experiments, stim-
ulation hampered the degradation of PrP
Sc
. Moreover,
the stimulation seemed to support Rocky Mountain
Laboratory strain (RML)-PrP
Sc
conversion in J774
macrophages and BMDMs.
Results
Transient up-regulation of PrP
c
surface
expression in stimulated cells
PrP
c
surface expression is necessary for cellular prion
conversion and, in susceptible cell lines, the amount of

PrP may dictate the rate of de novo synthesis of PrP
Sc
.
To verify this in activated phagocytic cells, we stimu-
lated J774 and BV-2 cells with LPS and CpG-ODN
for 4 h. As a control, cells were treated with nonstimu-
lating GpC-ODN or left untreated. Successful stimula-
tion was confirmed by the measurement of tumour
necrosis factor-a (TNF-a) secretion (data not shown).
Zero, 6, 12, 18 and 24 h after removing the stimuli,
cell surface PrP
c
was measured by fluorescence-associ-
ated cell sorting (FACS) analysis (Fig. 1). The mean
fluorescence value of the control cells was set as one
and the values of treated cells were expressed as x-fold
in relation to the control fluorescence value. In BV-2
cells (top panel), the surface expression of PrP
c
was
significantly increased 12 h after LPS stimulation
(2.1-fold increase). CpG-ODN-stimulated cells reacted
similarly, although the expression was only 1.5-fold
increased after 12 h. PrP
c
levels in GpC-ODN-treated
cells were comparable with those in untreated control
cells. In J774 cells (middle panel), the shift was even
more pronounced; 12 h after stimulation, the amount
of surface PrP

c
in CpG-ODN- and LPS-treated cells
was increased by 2.7- and 2.4-fold, respectively. In
both cell lines, PrP
c
levels decreased at the 18 and 24 h
time points. Similar to the cell lines, PrP
c
expression
levels of BMDMs were analysed at 0 and 12 h after
stimulation (bottom panel). A significant increase was
observed after 12 h in both LPS- and CpG-ODN-trea-
ted cells (1.8- and 1.5-fold, respectively). Quantitative
RT-PCR experiments revealed that the amount of PrP
mRNA was not affected by stimulation (data not
shown).
In summary, we found that, on stimulation of BV-2,
J774 and BMDM cells the surface expression of PrP
c
increased transiently. This was not caused by an aug-
mented transcription rate of the prnp gene.
Stimulation of BV-2, J774 and primary
macrophages influences their response
to prion challenge
To determine the effects of stimulation and subsequent
PrP
c
up-regulation on primary prion infection, BV-2
S. Gilch et al. Prion clearance in innate immune cells
FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS 5835

and J774 cells were treated for 4 h with LPS, CpG-
ODN, GpC-ODN, or left untreated. After removing
the stimulatory agents, cells were incubated with RML
brain homogenate for 24 h (Fig. 2). The cultures were
then washed extensively with phosphate-buffered saline
(NaCl ⁄ P
i
) and either lysed immediately (0 h) or after
further cultivation for 24 and 48 h without brain
homogenate. All cells exhibited similar growth, inde-
pendent of stimulation. Proteinase K (PK)-digested
lysates were subjected to detergent solubility assay for
separation of PrP
Sc
partitioning in the pellet fraction.
In order to ensure comparable amounts of PrP
Sc
detected in the immunoblot, the entire pellet fraction
of each time point was loaded. Thereby, the absolute
PrP
Sc
amount was monitored over the duration of the
experiment. In BV-2 cells (Fig. 2A), almost equal
amounts of RML-PrP
Sc
were found in pellets of cell
lysates immediately after prion challenge, independent
of the stimulation state of the cells (lanes 1–4). After
24 h, the RML-PrP
Sc

signal was reduced in lysates
from untreated (to  30%) and GpC-ODN-treated
( 50%) control cells (lanes 5 and 8). In cultures stim-
ulated with LPS (lane 6), a moderate decrease (to
21
30
1 2 3 4 5 6 7 8 9 10 11 12
co
co
LPS
CpG
GpC
LPS
CpG
GpC
co
LPS
CpG
GpC
co
co
LPS
CpG
GpC
LPS
CpG
GpC
co
LPS
CpG

GpC
0 h 24 h 48 h
BV-2
21
30
1 2 3 4 5 6 7 8 9 10 1112
0 h 24 h 48 h
J774
RML
RML
A
B
Fig. 2. Response of different cell types to infection with RML pri-
ons. (A) BV-2 microglial cells were stimulated for 4 h with LPS,
CpG-ODN, GpC-ODN, or left unstimulated (co) as indicated, and
were subsequently treated with RML-infected brain homogenate
for 24 h. The cells were then either lysed directly (0 h; lanes 1–4)
or after further cultivation (24 h, lanes 5–8; 48 h, lanes 9–12). Cell
lysates, representing equal amounts of viable cells, were subjected
to PK digestion and ultracentrifugation. Pellet fractions were analy-
sed by immunoblot using the monoclonal antibody 4H11. (B) A sim-
ilar analysis as in (A), performed with J774 murine macrophages.
Pellets of PK-digested and ultracentrifuged cell lysates were analy-
sed by immunoblot. PrP-specific bands were detected with the
monoclonal antibody 4H11.
ns ns
ns ns
*** *
*** *
*** *

ns *
ns ns
* **
* **
* **
ns ns
*** ***
ns ns
ns ns
*** *
*** *
*** *
ns *
ns ns
* **
* **
* **
ns ns
*** ***
BV-2
2,5
1,5
arbitrary units
0,5
0
0
CO
LPS CpG
GpC
CO

LPS CpG
GpC
CO
LPS CpG
GpC
612
hours
18 24
0612
hours
012
hours
18 24
2
1
3,5
2,5
1,5
arbitrary units
0,5
0
2
3
1
1,5
arbitrary units
0,5
0
2
1

J774
BMDM
Fig. 1. Kinetics of surface PrP
c
expression after stimulation of BV-2
microglial cells (top panel), J774 macrophages (middle panel) and
BMDMs (bottom panel) for 4 h with LPS, CpG-ODN, GpC-ODN, or
left unstimulated. Surface FACS analysis was performed in tripli-
cate after 0, 6, 12, 18 and 24 h following stimulation for BV-2 and
J774 (antibody against PrP A7) and after 0 and 12 h for BMDMs
(antibody against PrP 12F10). The average of the mean fluores-
cence intensity is shown and is expressed as an x-fold increase
relative to unstimulated control cells (value ¼ 1). Bars indicate
standard deviation. Statistical significance is indicated: ns, not
significant; *P < 0.05; **P < 0.005; ***P < 0.001.
Prion clearance in innate immune cells S. Gilch et al.
5836 FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS
 70%) was observed; in CpG-ODN-treated cells
(lane 7), the RML-PrP
Sc
signal was barely diminished.
Only in CpG-ODN-stimulated cells was a faint PrP
Sc
signal still detectable after 48 h.
In J774 cell lysates (Fig. 2B), a similar pattern, with
similar PrP
Sc
amounts in all cell lysates, was found at
the 0 h time point. Surprisingly, after 24 h, notably
without brain homogenate contained in the culture

medium, the RML-PrP
Sc
signal, particularly in LPS-
treated cells (lane 6) and, after 48 h, also in CpG-
ODN-treated cells (lane 11) was increased ( 1.8- and
1.3-fold, respectively) relative to the baseline signal
directly after infection (lanes 2 and 3). This finding
was reproducible and was not the case if nonstimulato-
ry LPS (data not shown) or GpC-ODN (lane 8 and
12) was applied. In LPS-stimulated samples, a pro-
nounced signal for RML-PrP
Sc
was still detectable
after 48 h (lane 10), whereas, in control and GpC-
ODN-treated cells, the signal again decreased. Five
days after infection, RML-PrP
Sc
was undetectable in
all cells (data not shown). To ensure that LPS and
CpG-ODN effects are caused by the activation of cells
via toll-like receptors (TLRs), N2a cells, which could
not be stimulated with LPS and CpG-ODN, were trea-
ted similarly to macrophages and microglial cells. No
LPS- or CpG-ODN-specific alterations in the PrP
Sc
signals were observed after the different time points
(data not shown).
According to the procedure described above, we
attempted to verify these results using 22L prions
(Fig. 3). In BV-2 cells, a strong PrP

Sc
signal and simi-
lar amounts of 22L-PrP
Sc
were detected on lysis
directly after incubation with 22L brain homogenate
(Fig. 3A; 0 h). After 24 h, a weak 22L-PrP
Sc
signal
was seen only in CpG-ODN-stimulated cells (lane 7).
After 48 h, no 22L-PrP
Sc
was detectable. J774 cells
showed a completely different picture (Fig. 3B).
Immediately after infection (0 h), large amounts of
22L-PrP
Sc
were detected in all cell lysates. By contrast
with the rapid disappearance of 22L-PrP
Sc
in BV-2
cells, in J774 cells, 22L-PrP
Sc
signals were completely
absent only after observation for 7 days. Of note, the
amount of 22L-PrP
Sc
found in these cells was only
slightly affected by stimulation, and the increase in
PrP

Sc
that was observed with RML prions was not
evident.
To support the relevance of the findings described
above, primary mouse BMDMs were prepared. Similar
to the cell lines, they were stimulated and incubated
with 22L or RML brain homogenate for 24 h. Cells
were lysed either immediately, or 24 or 48 h after
infection. PK-digested pellet fractions obtained by
detergent solubility assay were analysed by immuno-
21
30
1 2 3 4 5 6 7 8 9 10 11 12
c
o
LPS
CpG
GpC
c
o
LPS
CpG
GpC
c
o
LPS
CpG
GpC
0 h 24 h 48 h
BV-2

21
30
1 2 3 4 5 6 7 8 9 101112 13 14 15 16
c
o
LPS
CpG
GpC
c
o
LPS
CpG
GpC
c
o
LPS
CpG
GpC
0 d 2 d 5 d
J774
22L
22L
c
o
LPS
CpG
GpC
7 d
A
B

Fig. 3. Infection of BV-2 and J774 with 22L prions. (A) After stimu-
lation (co, LPS, CpG-ODN, GpC-ODN), BV-2 cells were incubated
for 24 h with brain homogenate derived from mice infected with
prion strain 22L. Lysates after different time points as indicated (0,
24, 48 h) were digested with PK, ultracentrifuged and the pellet
fractions were subjected to immunoblot analysis. For the detection
of PrP-specific bands, the monoclonal antibody 4H11 was used. (B)
J774 macrophages were treated as described in (A). After PK
digestion and ultracentrifugation of cell lysates prepared after the
different time points (0, 2, 5 and 7 days after infection), pellet frac-
tions were analysed by immunoblot using the monoclonal antibody
4H11.
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12
21
30
21
30
c
o
LPS
CpG
GpC
c
o
LPS
CpG
GpC
c
o

LPS
CpG
GpC
0 h 24 h 48 h
22L
c
o
LPS
CpG
GpC
c
o
LPS
CpG
GpC
c
o
LPS
CpG
GpC
0 h 24 h 48 h
RML
A
B
Fig. 4. Prion infection of BMDMs. (A) BMDMs were stimulated or
not as indicated for 4 h. Then, 22L brain homogenate was added
for 24 h. After washing the cells, they were lysed immediately
(0 h) or 24 and 48 h later, respectively. PK-digested pellet fractions
were analysed by immunoblot with monoclonal antibody 4H11. (B)
Identical experiment as in (A). RML brain homogenate was used

for infection.
S. Gilch et al. Prion clearance in innate immune cells
FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS 5837
blot. The entire pellet fraction was loaded to ensure
comparable conditions (Fig. 4). Like J774 macrophag-
es, BMDMs degraded 22L prions quite slowly without
an obvious influence of stimulation. By contrast, on
RML infection, an increase was observed in PrP
Sc
in
LPS-stimulated cells ( 1.4-fold) after 24 h incubation
without brain homogenate, and a slight increase in
CpG-ODN-stimulated cells.
PrP
Sc
accumulates intracellularly in macrophages
and microglial cells before degradation
To ascertain that J774 and BV-2 cells effectively inter-
nalize PrP
Sc
, indirect immunofluorescence assays under
conditions specific for the detection of PrP
Sc
[44] and
confocal microscopy were performed on stimulation
and infection with RML brain homogenate (Fig. 5). In
BV-2 J774
n. i.
co
CpG-ODN

LPS
Fig. 5. PrP
Sc
is located intracellularly in J774
and BV-2 cells. BV-2 (left panel) and J774
(right panel) cells were activated for 4 h or
left untreated (co, LPS, CpG), and then incu-
bated for 24 h with RML-infected brain
homogenate (co, LPS, CpG) or with unin-
fected brain homogenate (not infected, n.i.).
An immunofluorescence assay was per-
formed, including a denaturation step with
guanidinium hydrochloride (6
M), to allow
the specific detection of PrP
Sc
using the
monoclonal antibody 4H11.
Prion clearance in innate immune cells S. Gilch et al.
5838 FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS
cells treated with uninfected brain homogenate as
a control (n.i.), no specific fluorescence could be
detected, confirming that PrP
c
was not recognized
under our experimental conditions. In all samples
exposed to RML-infected brain homogenate (control,
CpG-ODN-treated, LPS-treated), specific intracellular
PrP
Sc

staining was found, independent of the activa-
tion state of the cells.
These results show that macrophages and microglial
cell lines are able to internalize and accumulate PrP
Sc
when exposed to prion-infected brain homogenate.
Transient prion conversion versus degradation
of PrP
Sc
in stimulated cells
In further experiments, we attempted to elucidate the
underlying mechanisms of the observations made in
the stimulation ⁄ infection experiments. To determine
whether the rapid reduction of 22L-PrP
Sc
in BV-2
cells was caused by effective degradation, we stimu-
lated BV-2 cells with the different reagents, followed
by infection with 22L prions. The cultures were then
rinsed with NaCl ⁄ P
i
, lysed directly, or cultivated for
a further 24 h in the presence or absence of NH
4
Cl
to inhibit endosomal ⁄ lysosomal proteases (Fig. 6A).
Pellet fractions, after detergent solubility assay of cell
lysates without PK digestion, were analysed by immu-
noblot. Of note, all samples contained N-terminally
truncated PrP

Sc
(PrP27–30), by contrast with the
brain homogenate used as inoculum in which mainly
full-length PrP
Sc
was found (Fig. 6B). When NH
4
Cl
was added to the cells, 22L-PrP
Sc
was detectable in
all samples, by contrast with cultures without NH
4
Cl.
The most prominent bands were found in cell lysates
of CpG-ODN-stimulated cells, with and without
NH
4
Cl treatment. These data indicate that, in micro-
glial cells, PrP
Sc
is rapidly degraded in acidic vesicles,
and that CpG-ODN treatment interferes with proteo-
lysis.
We assumed that the increased RML-PrP
Sc
signal in
stimulated J774 macrophages could be the result of
transient de novo generation of PrP
Sc

. To support this,
we stimulated J774 cells as indicated and incubated
them with RML brain homogenate. Cells were lysed
directly, or incubated for a further 24 h in culture
medium either with or without suramin (Fig. 6C). By
the addition of suramin to the cells, de novo synthesis
of PrP
Sc
is completely inhibited [45,46]. Pellet fractions
of cell lysates without PK digestion were tested by
immunoblot for their RML-PrP
Sc
content. Directly
after infection, all lysates contained similar amounts of
N-terminally truncated PrP
Sc
(PrP27–30). Without sur-
amin, the signal in LPS- and CpG-ODN-treated cells
was enhanced after 24 h. By contrast, when suramin
was added to the cells (lanes 10 and 11), the signals in
all lysates remained equal or even diminished relative
21
30
1 2 3 4 5 6 7 8 9 10 11 12
c
o
LPS
CpG
GpC
c

o
LPS
CpG
GpC
c
o
LPS
CpG
GpC
0 h
24 h
24 h
-NH
4
Cl
+ NH
4
Cl
BV-2
22L
1 2 3 4 5 6 7 8 9 10 11 12
21
30
c
o
LPS
CpG
GpC
c
o

LPS
CpG
GpC
co
LPS
CpG
GpC
co
LPS
CpG
GpC
c
o
LPS
CpG
GpC
0 h
24 h
24 h
- Sur + Sur
J774
RML
21
30
- - + + PK
1 2 3 4
22L
RML
RML
22L

A
B
C
Fig. 6. Principles underlying the observed effects. (A) BV-2 cells
were stimulated for 4 h as indicated (co, LPS, CpG, GpC), infected
for 24 h with 22L prions and lysed either directly (0 h, lanes 1–4)
or cultivated for another 24 h in culture medium in the absence
(– NH
4
Cl; lanes 5–8) or presence (+ NH
4
Cl; lanes 9–12) of ammo-
nium chloride. All cell lysates (– PK) were ultracentrifuged. Pellet
fractions were analysed by immunoblot. PrP-specific signals were
detected with the monoclonal antibody 4H11. (B) An aliquot of
RML- (lanes 1 and 3) or 22L- (lanes 2 and 4) infected brain homo-
genate was analysed by immunoblot without (lanes 1 and 2) or after
(lanes 3 and 4) PK digestion. For the detection of specific signals,
the monoclonal antibody 4H11 was used. (C) Following stimulation
(co, LPS, CpG, GpC) for 4 h, J774 macrophages were treated for
24 h with RML-infected brain homogenate. After removal, cells
were lysed immediately (0 h, lanes 1–4) or after further cultivation
for 24 h in the presence (+ Sur; 200 lgÆmL
)1
; lanes 9–12) or
absence (– Sur; lanes 5–8) of suramin. Pellet fractions of the
ultracentrifuged cell lysates were subjected to immunoblot, and PrP-
specific bands were visualized with the monoclonal antibody 4H11.
S. Gilch et al. Prion clearance in innate immune cells
FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS 5839

to the 0 h time point. This decrease indicates that the
effects observed on suramin treatment are not caused
by the potential inhibition of lysosomal degradation
by the compound.
Taken together, these results show that BV-2 cells
degrade PrP
Sc
in acidic compartments. J774 cells, if
infected with RML prions, may be able to transiently
synthesize PrP
Sc
. The generation of PrP27–30 demon-
strates the immediate N-terminal truncation of PrP
Sc
after phagocytosis.
Discussion
The aim of our study was to investigate the impact
of the stimulation of macrophages and microglial cells
by LPS or CpG on PrP
c
expression and their han-
dling of prion-infected brain material. We chose the
cell line J774, a differentiated murine macrophage-like
cell line exhibiting several features of primary macro-
phages, e.g. expression of Fc-receptors and a capabil-
ity of antigen presentation [47]. The cell line BV-2
exhibits most of the morphological, phenotypical and
functional properties described for freshly isolated
microglial cells [48]. To support the relevance of our
findings, key experiments were confirmed with pri-

mary BMDMs.
LPS- and CpG-ODN-induced PrP
c
up-regulation
does not alter PrP
Sc
uptake
Using FACS analysis, we found that, in all cells, sur-
face PrP
c
expression was significantly up-regulated
12 h after stimulation with LPS or CpG-ODN. The
PrP
c
levels then decreased again with similar kinetics.
When J774 and BV-2 cells were treated with prion-
infected brain homogenate, we initially assumed that
the stimulation of cells might result in a higher phago-
cytic and proteolytic activity [49]. However, this was
not the case. In a PrP
Sc
-specific immunofluorescence
assay [44], strong vesicular staining was found in both
cell lines, showing that PrP
Sc
is effectively internalized
by both cell lines, independent of stimulation and
of surface PrP
c
levels. In addition, both cell lines

harboured, almost exclusively, PrP
Sc
which was N-ter-
minally trimmed even without PK treatment, whereas
the inoculum mainly contained full-length PrP
Sc
(see
Fig. 6A,B), indicating partial proteolysis after phago-
cytosis. This led us to suggest that the processing of
PrP
Sc
in both cell lines occurs in two steps. First, full-
length PrP
Sc
is taken up by the cells and degradation
starts with the rapid digestion of the flexible N-termi-
nus, giving rise to PrP27–30. This material is handled
further in a cell type- and strain-specific manner.
Impaired degradation of PrP
Sc
in
CpG-ODN-stimulated microglial cells
In BV-2 cells, PrP
Sc
signals did not exceed the baseline
signal found immediately after infection. Here, CpG-
ODN, but not LPS, stimulation interfered with the
degradation of PrP
Sc
. A similar effect has been

described for skin dendritic cells [26]. Of note, in these
cells, the degradation of PrP
Sc
was hampered on LPS
activation, whereas the impact of CpG-ODN was not
addressed. For degradation, two main systems are
available for the cell: the cytosolic proteasomal degra-
dation machinery and the degradation in endosomal ⁄
lysosomal compartments. Arguing that phagocytosed
material is most probably subjected to lysosomal deg-
radation, we were able to confirm this by the inhibi-
tion of PrP
Sc
degradation with NH
4
Cl. The difference
between LPS and CpG-ODN treatment may be a
result of differences in the downstream signalling of
TLR4 and TLR9, through which different genes may
be activated [50]. In any case, our data do not support
the described putative protective role of CpG-ODN
application against prion disease [51], which is proba-
bly mainly caused by an altered spleen architecture
induced by stimulation and by the lack of cell types
supporting peripheral prion replication [52].
Does LPS stimulation support the transient
propagation of RML-PrP
Sc
in macrophages?
The results in J774 and BMDM cells were rather dif-

ferent to those in BV-2 cells. 22L prions were degraded
much more slowly than in BV-2 cells. Possibly, macro-
phages have the ability to store antigens, as has been
described for splenic dendritic cells, which then directly
interact with B-lymphocytes to trigger antibody pro-
duction [53]. In addition, the proteolytic capacity of
different cell types can influence the degradation kinet-
ics of various prion strains. The increase in the RML-
PrP
Sc
signal, particularly in LPS-stimulated J774 and
BMDM cells, was quite unexpected, and gives rise to
the hypothesis that these cells are able to transiently
convert RML-PrP
Sc
. It is worth noting that J774 and
primary BMDM cells both showed the same effect. As
the expression levels of PrP
c
, and therefore also of
newly converted PrP
Sc
, were below the detection limit
of both immunoblot and metabolic labelling followed
by radio-immunoprecipitation (data not shown), even
after stimulation, we employed the compound suramin
to inhibit the de novo synthesis of PrP
Sc
[45,46].
Indeed, the increase in RML-PrP

Sc
in J774 cells was
thereby prevented, which strengthens the hypothesis of
transient PrP
Sc
propagation, at least in a transient and
Prion clearance in innate immune cells S. Gilch et al.
5840 FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS
strain-dependent manner Therefore, this is the first
report to show that cultured macrophages may be able
to propagate PrP
Sc
. This was only the case in stimu-
lated cells, which can be explained by the increased
surface PrP
c
levels. Nevertheless, there is no correla-
tion between the increase in the level of PrP
c
and the
amount of possibly converted RML-PrP
Sc
. If this were
the case, one would expect a more pronounced RML-
PrP
Sc
increase in CpG-ODN-stimulated cells, as FACS
data indicate higher surface PrP
c
levels. It should be

noted that these data do not implicitly indicate that
macrophage cell lines are infectable as, on transient
formation of PrP
Sc
, persistent infection is not necessar-
ily established in cultured cells [54]. Evidence for prion
replication in macrophages is provided in vivo, as, in
mice lacking FDCs, lymph node prion replication is
associated with macrophage subsets [20]. In J774 cells,
RML-PrP
Sc
was finally degraded, and 5 days after
infection no RML-PrP
Sc
was detectable in stimulated
cells by immunoblot analysis (data not shown). These
results indicate a scenario in which, on coinfection
with prions and bacteria or viruses delivering agonists
of TLR signalling, uptake of PrP
Sc
by macrophages is,
at least for a certain time frame, no longer beneficial
for the clearance of prions, in line with an early report
on the increased susceptibility of mice to scrapie on
stimulation with phytohaemagglutinin [55]. Recruit-
ment of immune cells to sites of chronic inflammation
in prion-infected animals can alter the organ tropism
of prions [56–58], and the activation of these immune
cells may also facilitate prion replication in peripheral
organs usually not prone to the generation of PrP

Sc
.
In summary, our data do not support a solely pro-
tective role of the stimulation of macrophages and
microglial cells in primary prion infection scenarios.
Stimulation and subsequent PrP
c
up-regulation do not
enhance PrP
Sc
uptake, but may disturb the cellular bal-
ance between degradation and propagation.
Experimental procedures
Reagents
PK and Pefabloc proteinase inhibitor were obtained from
Roche, Mannheim, Germany. LPS from Escherichia coli
was obtained from Sigma, Deisenhofen, Germany. CpG
and GpC motif-containing oligodeoxynucleotides (CpG-
and GpC-ODN 1668 and 1720, respectively) were obtained
from TIB Molbiol (Berlin, Germany). Immunoblotting was
performed using the enhanced chemiluminescence blotting
technique (ECL plus) from Pharmacia (Freiburg, Germany).
A7 and 4H11 antibodies against PrP have been described
previously [59]. Monoclonal antibody against PrP 12F10
was purchased from Antiko
¨
rper Online, GmbH, Aachen,
Germany. The antibody against CD16 ⁄ CD32 was obtained
from BD Pharmingen (Heidelberg, Germany). Fluorescein
isothiocyanate (FITC)- and rhodamine-conjugated second-

ary antibodies were obtained from Dako or Dianova
(Hamburg, Germany). Cell culture media and solutions
were obtained from Gibco BRL (Karlsruhe, Germany).
Cell culture, stimulation and treatment of cells
The murine macrophage cell line J774 (ATCC TIB 67) and
the microglial cell line BV-2 [48] were kept in RPMI1680
medium supplemented with 7.5% fetal bovine serum (ultra-
low endotoxin), mercaptoethanol (50 lm) and antibiotics.
BMDMs were prepared from C57Bl ⁄ 6 mice. Bone marrow
cells were incubated overnight with macrophage colony-
stimulating factor containing L929 cell culture supernatant.
Then nonadherent cells were re-plated and differentiated for
7 days. Adherent cells were used for further analysis [60].
For stimulation, CpG-ODN and GpC-ODN were added at
a concentration of 1 lm, and LPS at 1 lgÆmL
)1
, for 4 h.
Medium was collected, centrifuged for 5 min at 600 g and
stored at ) 20 °C until testing for TNF-a secretion by
ELISA (R & D Developments, Minneapolis, MN, USA).
Suramin was dissolved in 0.9% NaCl at a stock concentra-
tion of 200 mgÆmL
)1
and added to the cells at a concentra-
tion of 200 lgÆmL
)1
for 24 h. Ammonium chloride (NH
4
Cl)
was applied at a concentration of 50 lm for 24 h.

Mode of transient prion infection
For transient prion infection, the mouse-adapted scrapie
strains RML and 22L were used. To prepare brain homo-
genates (10% w ⁄ v), infected brains from CD-1 (RML) and
C57Bl ⁄ 6 (22L) mice were homogenized in NaCl ⁄ P
i
. After
stimulation of cells for 4 h, the stimuli were removed and
brain homogenate was added to the cells at a 1 : 10 dilution
in culture medium (final concentration of 1%) for 24 h. For
stimulation and treatment with brain homogenate, cells were
kept on 10 cm dishes in order to ensure equal stimulation
and infection conditions. After washing these cells with
NaCl ⁄ P
i
, they were divided equally on 6 cm dishes for the
various chase points. One part of the cells was lysed imme-
diately after removal of the brain material, and was denoted
as the 0 h time point. All lysates (with and without PK
digestion) were subjected to a solubility assay. The entire
pellet fraction of each time point was analysed by immuno-
blot to allow the comparison of PrP
Sc
amounts.
Cell lysis, PK analysis and immunoblot
Confluent cell cultures were washed twice in cold NaCl ⁄ P
i
and lysed in 1 mL cold lysis buffer (10 mm Tris ⁄ HCl,
pH 7.5, 100 mm NaCl, 10 mm EDTA, 0.5% Triton X-100,
S. Gilch et al. Prion clearance in innate immune cells

FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS 5841
0.5% deoxycholate) for 10 min. After centrifugation at
10 000 g for 1 min, the supernatant samples were split
between those without and with PK digestion (20 lgÆmL
)1
for 30 min at 37 °C). Digestion was stopped with Pefabloc
and samples were subjected to detergent solubility assay.
After the addition of sample buffer to the re-suspended pel-
let fractions after detergent solubility assay and boiling for
5 min, an aliquot was analysed by 12.5% PAGE. For Wes-
tern blot analysis, the proteins were electrotransferred to
poly(vinylidene difluoride) membranes (Pharmacia). The
membrane was blocked with 5% nonfat dry milk in NaCl ⁄
Tris T (0.05% Tween 20, 100 mm NaCl, 10 mm Tris ⁄ HCl,
pH 7.8), incubated overnight with the primary antibody at
4 °C and stained using the enhanced chemiluminescence
blotting (ECL plus) kit from Pharmacia.
Detergent solubility assay
Cells were lysed in lysis buffer as described for immunoblot
analysis. Postnuclear cell lysates (± PK) were supple-
mented with Pefabloc and N-lauryl sarcosine (1%), and
ultracentrifuged in a Beckman (Krefeld, Germany) TL-100
table ultracentrifuge for 1 h at 100 000 g using a TLA-45
rotor at 4 °C). Pellet fractions were re-suspended in 20 lL
of TNE (50 lm Tris/HCl, 150 mm NaCl, 5 mm EDTA, pH
7.4) and analysed by immunoblot.
FACS analysis
For the analysis of surface protein expression, cells were
suspended in FACS buffer (2.5% fetal bovine serum and
0.05% NaN

3
in NaCl ⁄ P
i
) and incubated for 5 min on ice.
After centrifugation, Fc-receptors were blocked by incuba-
tion of cells with antibody against CD16 ⁄ CD32 (1 : 100;
BD Pharmingen) for 30 min on ice. After three washes with
FACS buffer, primary anti-PrP antibodies (A7 or 12F10)
were added in a 1 : 100 dilution in FACS buffer for 45 min
on ice, washed three times in FACS buffer, and the second-
ary antibody (FITC-labelled, 1 : 100) was added and incu-
bated for another 45 min. After the last wash, cells were
re-suspended in FACS buffer containing 7-amino-actino-
mycin D (BD Pharmingen). FACS analysis was performed
in a Coulter Epics XL MCL apparatus (Beckman Coulter,
Krefeld, Germany). Statistical analysis was performed by
comparing differences between LPS or CpG-ODN stimu-
lation with GpC-ODN-treated cells in an unpaired two-
tailed t-test using graphpadprism software.
PrP
Sc
-specific indirect immunofluorescence assay
and confocal laser scanning microscopy
Cells were plated on glass cover slips (Marienfeld, Ger-
many) at low density. They were washed twice in cold
NaCl ⁄ P
i
and fixed in 4% paraformaldehyde for 30 min at
room temperature. After sequential treatment with NH
4

Cl
(50 mm in 20 mm glycine), Triton X-100 (0.3%), guanidini-
um hydrochloride (6 m) and gelatine (0.2%) for 10 min
each at room temperature and blocking of Fc-receptors, the
first antisera were added at 1 : 100 (e.g. 4H11) in NaCl ⁄ P
i
and incubated for 30 min at room temperature. After three
washes in NaCl ⁄ P
i
, FITC- or rhodamine-conjugated sec-
ondary antisera (1 : 100 dilution in NaCl ⁄ P
i
) were used and
immunostaining was accomplished according to standard
procedures. Slides were mounted in Permafluor Mounting
Medium (Beckman Coulter). Confocal laser scanning
microscopy was performed using a Zeiss LSM510 Confocal
System (Zeiss, Go
¨
ttingen, Germany).
Acknowledgements
We are grateful to Professors M. Groschup and H.
Kretzschmar for providing infected mouse brains. This
work was supported by SFB-576 (project B12), SFB-
596 (project A8 and Z1), DFG (Scha594 ⁄ 3-4) and the
EU NoE Neuroprion.
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