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 pri
Trang 1in macrophage and microglial cells
Sabine Gilch1, Frank Schmitz2, Yasmine Aguib1, Claudia Kehler1, Sigrid Bu¨low1, Stefan Bauer3, Elisabeth Kremmer4and Hermann M Scha¨tzl1
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 PrPc, designated PrPSc, which appears to be
the causative agent of disease [1–4] PrPcis 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 PrPcis 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 PrPcis mainly a-helical [7] During prion conversion, it interacts with PrPSc 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: schaetzl@lrz.tum.de
(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 (PrPc) is up-regu-lated on stimulation of immune cells, also indicating the functional impor-tance of PrPc in the immune system The aim of our study was to investigate the impact of cytosine-phosphate-guanosine- and lipopolysac-charide-induced PrPc up-regulation on the uptake and processing of the pathological prion protein (PrPSc) 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 PrPSc Interestingly, stimulation led either to a transient increase in the level of PrPScrelative to unstimulated cells or to a deceler-ated degradation of PrPSc 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 PrPc 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 PrPSc
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; PrPc, cellular prion protein; PrPSc, 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.
Trang 2[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 PrPSc [23–25] and, also, can
transport PrPSc, 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 PrPc has not yet been
clarified Some evidence indicates a functional role of
PrPc in the immune system The expression of PrPc
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 PrPc 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 PrPc up-regulation in macrophage
or microglial cell lines and primary macrophages on the
processing of PrPSc 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 PrPcof 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 PrPSc Moreover,
the stimulation seemed to support Rocky Mountain
Laboratory strain (RML)-PrPSc conversion in J774 macrophages and BMDMs
Results
Transient up-regulation of PrPcsurface expression in stimulated cells
PrPc 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 PrPSc
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 PrPc 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 PrPc 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 PrPc 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 PrPc in CpG-ODN- and LPS-treated cells was increased by 2.7- and 2.4-fold, respectively In both cell lines, PrPclevels decreased at the 18 and 24 h time points Similar to the cell lines, PrPc 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 PrPc 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 PrPc up-regulation on primary prion infection, BV-2
Trang 3and J774 cells were treated for 4 h with LPS,
CpG-ODN, GpC-CpG-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⁄ Pi) 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 PrPSc partitioning in the pellet fraction
In order to ensure comparable amounts of PrPSc detected in the immunoblot, the entire pellet fraction
of each time point was loaded Thereby, the absolute PrPScamount was monitored over the duration of the experiment In BV-2 cells (Fig 2A), almost equal amounts of RML-PrPSc 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-PrPSc 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 LPS CpG GpC co LPS CpG GpC co LPS CpG GpC
co LPS CpG GpC co LPS CpG GpC co LPS CpG GpC
BV-2
21 30
1 2 3 4 5 6 7 8 9 10 1112
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
0,5
0
0
CO LPS CpG GpC
CO LPS CpG GpC
CO LPS CpG GpC
hours
hours
hours
2
1
3,5
2,5
1,5
0,5
0
2
3
1
1,5
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.
Trang 470%) was observed; in CpG-ODN-treated cells
(lane 7), the RML-PrPScsignal was barely diminished
Only in CpG-ODN-stimulated cells was a faint PrPSc
signal still detectable after 48 h
In J774 cell lysates (Fig 2B), a similar pattern, with
similar PrPSc 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-PrPSc 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-PrPSc 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-PrPSc 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 PrPSc
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 PrPSc signal and
simi-lar amounts of 22L-PrPSc were detected on lysis
directly after incubation with 22L brain homogenate
(Fig 3A; 0 h) After 24 h, a weak 22L-PrPSc signal
was seen only in CpG-ODN-stimulated cells (lane 7)
After 48 h, no 22L-PrPSc was detectable J774 cells
showed a completely different picture (Fig 3B)
Immediately after infection (0 h), large amounts of
22L-PrPScwere detected in all cell lysates By contrast
with the rapid disappearance of 22L-PrPSc in BV-2
cells, in J774 cells, 22L-PrPSc signals were completely
absent only after observation for 7 days Of note, the
amount of 22L-PrPSc found in these cells was only
slightly affected by stimulation, and the increase in
PrPSc 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
co LPS CpG GpC co 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 101112 13 14 15 16
co LPS CpG GpC co LPS CpG GpC co LPS CpG GpC
0 d 2 d 5 d
J774
22L
22L
co 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
co LPS CpG GpC co LPS CpG GpC co LPS CpG GpC
0 h 24 h 48 h
22L
co LPS CpG GpC co LPS CpG GpC co 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.
Trang 5blot 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 PrPSc in
LPS-stimulated cells ( 1.4-fold) after 24 h incubation
without brain homogenate, and a slight increase in
CpG-ODN-stimulated cells
PrPSc accumulates intracellularly in macrophages and microglial cells before degradation
To ascertain that J774 and BV-2 cells effectively inter-nalize PrPSc, indirect immunofluorescence assays under conditions specific for the detection of PrPSc [44] and confocal microscopy were performed on stimulation and infection with RML brain homogenate (Fig 5) In
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 inunin-fected, n.i.).
An immunofluorescence assay was per-formed, including a denaturation step with guanidinium hydrochloride (6 M ), to allow the specific detection of PrPScusing the monoclonal antibody 4H11.
Trang 6cells treated with uninfected brain homogenate as
a control (n.i.), no specific fluorescence could be
detected, confirming that PrPc was not recognized
under our experimental conditions In all samples
exposed to RML-infected brain homogenate (control,
CpG-ODN-treated, LPS-treated), specific intracellular
PrPSc 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 PrPSc
when exposed to prion-infected brain homogenate
Transient prion conversion versus degradation
of PrPScin 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-PrPSc 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⁄ Pi, lysed directly, or cultivated for
a further 24 h in the presence or absence of NH4Cl
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 PrPSc (PrP27–30), by contrast with the
brain homogenate used as inoculum in which mainly
full-length PrPSc was found (Fig 6B) When NH4Cl
was added to the cells, 22L-PrPSc was detectable in
all samples, by contrast with cultures without NH4Cl
The most prominent bands were found in cell lysates
of CpG-ODN-stimulated cells, with and without
NH4Cl treatment These data indicate that, in
micro-glial cells, PrPSc is rapidly degraded in acidic vesicles,
and that CpG-ODN treatment interferes with
proteo-lysis
We assumed that the increased RML-PrPScsignal in
stimulated J774 macrophages could be the result of
transient de novo generation of PrPSc 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 PrPScis completely inhibited [45,46] Pellet fractions
of cell lysates without PK digestion were tested by
immunoblot for their RML-PrPSc content Directly
after infection, all lysates contained similar amounts of
N-terminally truncated PrPSc(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
co LP
CpG GpC co LPS CpG GpC co LP
CpG GpC
0 h
24 h 24 h
- NH4Cl + NH
4Cl BV-2
22L
1 2 3 4 5 6 7 8 9 10 11 12 21
30
co LPS Cp
co LPS Cp
GpC co LPS Cp
co LPS Cp
GpC co LPS CpG GpC
0 h
24 h
24 h
- Sur + Sur J774
RML
21 30
- - + + PK
1 2 3 4
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 (– NH4Cl; lanes 5–8) or presence (+ NH4Cl; 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.
Trang 7to 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 PrPSc in acidic compartments J774 cells, if
infected with RML prions, may be able to transiently
synthesize PrPSc The generation of PrP27–30
demon-strates the immediate N-terminal truncation of PrPSc
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 PrPc 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 PrPcup-regulation
does not alter PrPScuptake
Using FACS analysis, we found that, in all cells,
sur-face PrPc expression was significantly up-regulated
12 h after stimulation with LPS or CpG-ODN The
PrPc 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 PrPSc-specific immunofluorescence
assay [44], strong vesicular staining was found in both
cell lines, showing that PrPSc is effectively internalized
by both cell lines, independent of stimulation and
of surface PrPc levels In addition, both cell lines
harboured, almost exclusively, PrPSc which was
N-ter-minally trimmed even without PK treatment, whereas
the inoculum mainly contained full-length PrPSc (see
Fig 6A,B), indicating partial proteolysis after
phago-cytosis This led us to suggest that the processing of
PrPScin both cell lines occurs in two steps First,
full-length PrPSc 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 PrPScin CpG-ODN-stimulated microglial cells
In BV-2 cells, PrPScsignals did not exceed the baseline signal found immediately after infection Here, CpG-ODN, but not LPS, stimulation interfered with the degradation of PrPSc A similar effect has been described for skin dendritic cells [26] Of note, in these cells, the degradation of PrPScwas 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 degradegra-dation 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 PrPScdegradation with NH4Cl 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-PrPScin 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-PrPSc 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-PrPSc It is worth noting that J774 and primary BMDM cells both showed the same effect As the expression levels of PrPc, and therefore also of newly converted PrPSc, 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 PrPSc [45,46] Indeed, the increase in RML-PrPSc in J774 cells was thereby prevented, which strengthens the hypothesis of transient PrPScpropagation, at least in a transient and
Trang 8strain-dependent manner Therefore, this is the first
report to show that cultured macrophages may be able
to propagate PrPSc This was only the case in
stimu-lated cells, which can be explained by the increased
surface PrPc levels Nevertheless, there is no
correla-tion between the increase in the level of PrPc and the
amount of possibly converted RML-PrPSc If this were
the case, one would expect a more pronounced
RML-PrPScincrease in CpG-ODN-stimulated cells, as FACS
data indicate higher surface PrPc levels It should be
noted that these data do not implicitly indicate that
macrophage cell lines are infectable as, on transient
formation of PrPSc, 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-PrPSc was finally degraded, and 5 days after
infection no RML-PrPSc 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 PrPScby 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 PrPSc
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 PrPc up-regulation do not
enhance PrPScuptake, 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 (CpG-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)1and added to the cells at a concentra-tion of 200 lgÆmL)1for 24 h Ammonium chloride (NH4Cl) 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 ⁄ Pi 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⁄ Pi, 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 PrPScamounts
Cell lysis, PK analysis and immunoblot Confluent cell cultures were washed twice in cold NaCl⁄ Pi
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,
Trang 90.5% 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 37C) 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
4C 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 4C) 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% NaN3 in NaCl⁄ Pi) 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
PrPSc-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⁄ Pi and fixed in 4% paraformaldehyde for 30 min at
room temperature After sequential treatment with NH4Cl (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⁄ Pi
and incubated for 30 min at room temperature After three washes in NaCl⁄ Pi, FITC- or rhodamine-conjugated sec-ondary antisera (1 : 100 dilution in NaCl⁄ Pi) 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 576 (project B12),
SFB-596 (project A8 and Z1), DFG (Scha594⁄ 3-4) and the
EU NoE Neuroprion
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