CpG and LPS can interfere negatively with prion clearance in macrophage and microglial cells Sabine Gilch1, Frank Schmitz2, Yasmine Aguib1, Claudia Kehler1, Sigrid Bulow1, Stefan Bauer3, ă Elisabeth Kremmer4 and Hermann M Schatzl1 ă Institute of Virology, Prion Research Group, Technical University of Munich, Germany Institute of Microbiology and Immunology, Technical University of Munich, Germany Institute of Immunology, Philipps-University Marburg, Germany GSF-National Research Centre for Environment and Health, Institute of Molecular Immunology, Munich, Germany Keywords innate immunity; prion; prion clearance; PAMP; toll-like receptor Correspondence H M Schatzl, Institute of Virology, Prion ă 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 July 2007, revised 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-regulated on stimulation of immune cells, also indicating the functional importance of PrPc in the immune system The aim of our study was to investigate the impact of cytosine-phosphate-guanosine- and lipopolysaccharide-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 PrPSc relative to unstimulated cells or to a decelerated 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 Prion diseases are fatal neurodegenerative disorders, including scrapie in sheep, bovine spongiform encephalopathy 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] PrPc is a glycoprotein 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 PrPc is crucial for the development of prion diseases, as mice ablated for the prnp gene not succumb to the disease [6] The structure of soluble PrPc is 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 aggregation [8,9] This probably occurs at the plasma membrane or in the early endocytic pathway, but the exact subcellular site of prion conversion has not been identified [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 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 5834 FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS S Gilch et al [13,14] On peripheral infection, a huge body of evidence points to the role of immune cells in the neuroinvasion process [15–18] Transport through epithelial colon cells in the presence of differentiated M-cells may enable prions to gain first access to the lymphoreticular 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 neuroinvasion is still controversial [15,22,25–27] By contrast, macrophages may be involved in the clearance of prions [28–31] Microglial cells are resident brain macrophages and become activated during the progression of prion disease [32] They contribute to the neurodegenerative 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 overexpressing 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 nonplasmacytoid dendritic cells, on activated T-cells or on interferon-c-treated monocytes [37–40] Immunization of mice with vesicular stomatitis virus led to an up-regulation 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 marrow-derived macrophages (BMDMs) On activation with cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODNs) or lipopolysaccharide (LPS), cells showed an up-regulation of PrPc of about twofold with similar kinetics There were distinct differences in the reaction to prion infection, but, in all experiments, stimulation hampered the degradation of PrPSc Moreover, the stimulation seemed to support Rocky Mountain Prion clearance in innate immune cells Laboratory strain (RML)-PrPSc conversion in J774 macrophages and BMDMs Results Transient up-regulation of PrPc surface 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 stimulated J774 and BV-2 cells with LPS and CpG-ODN for h As a control, cells were treated with nonstimulating GpC-ODN or left untreated Successful stimulation 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-associated 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, PrPc levels decreased at the 18 and 24 h time points Similar to the cell lines, PrPc expression levels of BMDMs were analysed at and 12 h after stimulation (bottom panel) A significant increase was observed after 12 h in both LPS- and CpG-ODN-treated 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 augmented 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 FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS 5835 Prion clearance in innate immune cells S Gilch et al BV-2 *** * arbitrary units 1,5 0h *** * *** * ns ns RML 21 10 11 12 CO LPS CpG GpC 12 hours 18 24 0h J774 3,5 * ** * ** ns ns 2,5 J774 B 24 h 48 h co LPS CpG GpC co LPS CpG GpC co LPS CpG GpC arbitrary units 48 h 30 0,5 30 RML * ** ns * 21 1,5 0,5 0 CO LPS CpG GpC 12 hours 18 24 BMDM arbitrary units 24 h co LPS CpG GpC co LPS CpG GpC co LPS CpG GpC ns ns BV-2 A 2,5 *** *** ns ns 1,5 0,5 0 CO LPS CpG hours 12 GpC Fig Kinetics of surface PrPc expression after stimulation of BV-2 microglial cells (top panel), J774 macrophages (middle panel) and BMDMs (bottom panel) for h with LPS, CpG-ODN, GpC-ODN, or left unstimulated Surface FACS analysis was performed in triplicate after 0, 6, 12, 18 and 24 h following stimulation for BV-2 and J774 (antibody against PrP A7) and after and 12 h for BMDMs (antibody against PrP 12F10) The average of the mean fluorescence 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 and J774 cells were treated for h with LPS, CpGODN, 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 5836 10 1112 Fig Response of different cell types to infection with RML prions (A) BV-2 microglial cells were stimulated for 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 analysed by immunoblot using the monoclonal antibody 4H11 (B) A similar analysis as in (A), performed with J774 murine macrophages Pellets of PK-digested and ultracentrifuged cell lysates were analysed by immunoblot PrP-specific bands were detected with the monoclonal antibody 4H11 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, independent 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 PrPSc amount 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 and 8) In cultures stimulated with LPS (lane 6), a moderate decrease (to FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS S Gilch et al BV-2 24 h A 48 h co LPS CpG GpC co LPS CpG GpC co LPS CpG GpC 0h 30 22L 21 10 11 12 J774 B 2d 7d 5d co LPS CpG GpC co LPS CpG GpC co LPS CpG GpC co LPS CpG GpC 0d 30 22L 21 101112 13 14 15 16 24 h 48 h co LPS CpG GpC co LPS CpG GpC A 0h co LPS CpG GpC Fig Infection of BV-2 and J774 with 22L prions (A) After stimulation (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, and days after infection), pellet fractions were analysed by immunoblot using the monoclonal antibody 4H11 30 22L 10 11 12 0h 24 h 48 h co LPS CpG GpC B co LPS CpG GpC 21 co LPS CpG GpC  70%) was observed; in CpG-ODN-treated cells (lane 7), the RML-PrPSc signal 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 h time point Surprisingly, after 24 h, notably without brain homogenate contained in the culture medium, the RML-PrPSc signal, particularly in LPStreated cells (lane 6) and, after 48 h, also in CpGODN-treated cells (lane 11) was increased ( 1.8- and 1.3-fold, respectively) relative to the baseline signal directly after infection (lanes and 3) This finding was reproducible and was not the case if nonstimulatory LPS (data not shown) or GpC-ODN (lane and 12) was applied In LPS-stimulated samples, a pronounced signal for RML-PrPSc was still detectable after 48 h (lane 10), whereas, in control and GpCODN-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 treated 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 similar amounts of 22L-PrPSc were detected on lysis directly after incubation with 22L brain homogenate (Fig 3A; 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-PrPSc were 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 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- Prion clearance in innate immune cells 30 RML 21 10 11 12 Fig Prion infection of BMDMs (A) BMDMs were stimulated or not as indicated for 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 FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS 5837 Prion clearance in innate immune cells S Gilch et al blot The entire pellet fraction was loaded to ensure comparable conditions (Fig 4) Like J774 macrophages, 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 BV-2 PrPSc accumulates intracellularly in macrophages and microglial cells before degradation To ascertain that J774 and BV-2 cells effectively internalize 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 J774 n i co CpG-ODN LPS 5838 Fig PrPSc is located intracellularly in J774 and BV-2 cells BV-2 (left panel) and J774 (right panel) cells were activated for h or left untreated (co, LPS, CpG), and then incubated for 24 h with RML-infected brain homogenate (co, LPS, CpG) or with uninfected brain homogenate (not infected, n.i.) An immunofluorescence assay was performed, including a denaturation step with guanidinium hydrochloride (6 M), to allow the specific detection of PrPSc using the monoclonal antibody 4H11 FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS S Gilch et al BV-2 0h 24 h - NH4Cl 24 h + NH4Cl LPS CpG GpC co LPS CpG GpC co LPS CpG GpC A co 30 22L 21 B Transient prion conversion versus degradation of PrPSc in stimulated cells 10 11 12 - - + + PK 30 21 24 h + Sur LPS CpG GpC C co 0h J774 24 h - Sur co LPS CpG GpC co LPS CpG GpC 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 stimulated 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 immunoblot 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 microglial cells, PrPSc is rapidly degraded in acidic vesicles, and that CpG-ODN treatment interferes with proteolysis We assumed that the increased RML-PrPSc signal 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 PrPSc is 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 suramin, the signal in LPS- and CpG-ODN-treated cells RML 22L RML 22L cells 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 activation 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 Prion clearance in innate immune cells 30 RML 21 10 11 12 Fig Principles underlying the observed effects (A) BV-2 cells were stimulated for 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 ammonium 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 and 3) or 22L- (lanes and 4) infected brain homogenate was analysed by immunoblot without (lanes and 2) or after (lanes and 4) PK digestion For the detection of specific signals, the monoclonal antibody 4H11 was used (C) Following stimulation (co, LPS, CpG, GpC) for 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 lgỈmL)1; lanes 9–12) or absence (– Sur; lanes 5–8) of suramin Pellet fractions of the ultracentrifuged cell lysates were subjected to immunoblot, and PrPspecific bands were visualized with the monoclonal antibody 4H11 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 FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS 5839 Prion clearance in innate immune cells S Gilch et al to the 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 demonstrates the immediate N-terminal truncation of PrPSc after phagocytosis Impaired degradation of PrPSc in CpG-ODN-stimulated microglial cells 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 handling of prion-infected brain material We chose the cell line J774, a differentiated murine macrophage-like cell line exhibiting several features of primary macrophages, e.g expression of Fc-receptors and a capability 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 primary BMDMs In BV-2 cells, PrPSc signals did not exceed the baseline signal found immediately after infection Here, CpGODN, 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 PrPSc 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 degradation machinery and the degradation in endosomal ⁄ lysosomal compartments Arguing that phagocytosed material is most probably subjected to lysosomal degradation, we were able to confirm this by the inhibition of PrPSc degradation 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 not support the described putative protective role of CpG-ODN application against prion disease [51], which is probably mainly caused by an altered spleen architecture induced by stimulation and by the lack of cell types supporting peripheral prion replication [52] LPS- and CpG-ODN-induced PrPc up-regulation does not alter PrPSc uptake Does LPS stimulation support the transient propagation of RML-PrPSc in macrophages? Using FACS analysis, we found that, in all cells, surface 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 prioninfected brain homogenate, we initially assumed that the stimulation of cells might result in a higher phagocytic 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-terminally trimmed even without PK treatment, whereas the inoculum mainly contained full-length PrPSc (see Fig 6A,B), indicating partial proteolysis after phagocytosis This led us to suggest that the processing of PrPSc in both cell lines occurs in two steps First, fulllength PrPSc is taken up by the cells and degradation starts with the rapid digestion of the flexible N-terminus, giving rise to PrP27–30 This material is handled further in a cell type- and strain-specific manner The results in J774 and BMDM cells were rather different to those in BV-2 cells 22L prions were degraded much more slowly than in BV-2 cells Possibly, macrophages have the ability to store antigens, as has been described for splenic dendritic cells, which then directly interact with B-lymphocytes to trigger antibody production [53] In addition, the proteolytic capacity of different cell types can influence the degradation kinetics of various prion strains The increase in the RMLPrPSc 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 PrPSc propagation, at least in a transient and Discussion 5840 FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS S Gilch et al strain-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 stimulated cells, which can be explained by the increased surface PrPc levels Nevertheless, there is no correlation 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 RMLPrPSc increase in CpG-ODN-stimulated cells, as FACS data indicate higher surface PrPc levels It should be noted that these data not implicitly indicate that macrophage cell lines are infectable as, on transient formation of PrPSc, persistent infection is not necessarily 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 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 PrPSc 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] Recruitment 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 not support a solely protective role of the stimulation of macrophages and microglial cells in primary prion infection scenarios Stimulation and subsequent PrPc up-regulation not enhance PrPSc uptake, but may disturb the cellular balance 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 (CpGand 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 Prion clearance in innate immune cells was purchased from Antikorper Online, GmbH, Aachen, ă Germany The antibody against CD16 ⁄ CD32 was obtained from BD Pharmingen (Heidelberg, Germany) Fluorescein isothiocyanate (FITC)- and rhodamine-conjugated secondary 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 (ultralow endotoxin), mercaptoethanol (50 lm) and antibiotics BMDMs were prepared from C57Bl ⁄ mice Bone marrow cells were incubated overnight with macrophage colonystimulating factor containing L929 cell culture supernatant Then nonadherent cells were re-plated and differentiated for days Adherent cells were used for further analysis [60] For stimulation, CpG-ODN and GpC-ODN were added at a concentration of lm, and LPS at lgỈmL)1, for h Medium was collected, centrifuged for 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 concentration of 200 mgỈmL)1 and added to the cells at a concentration of 200 lgỈmL)1 for 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 homogenates (10% w ⁄ v), infected brains from CD-1 (RML) and C57Bl ⁄ (22L) mice were homogenized in NaCl ⁄ Pi After stimulation of cells for h, the stimuli were removed and brain homogenate was added to the cells at a : 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 cm dishes for the various chase points One part of the cells was lysed immediately after removal of the brain material, and was denoted as the 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 immunoblot to allow the comparison of PrPSc amounts Cell lysis, PK analysis and immunoblot Confluent cell cultures were washed twice in cold NaCl ⁄ Pi and lysed in mL cold lysis buffer (10 mm Tris ⁄ HCl, pH 7.5, 100 mm NaCl, 10 mm EDTA, 0.5% Triton X-100, FEBS Journal 274 (2007) 5834–5844 ª 2007 The Authors Journal compilation ª 2007 FEBS 5841 Prion clearance in innate immune cells S Gilch et al 0.5% deoxycholate) for 10 After centrifugation at 10 000 g for min, the supernatant samples were split between those without and with PK digestion (20 lgỈmL)1 for 30 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 pellet fractions after detergent solubility assay and boiling for min, an aliquot was analysed by 12.5% PAGE For Western 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 °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 supplemented with Pefabloc and N-lauryl sarcosine (1%), and ultracentrifuged in a Beckman (Krefeld, Germany) TL-100 table ultracentrifuge for h at 100 000 g using a TLA-45 rotor at °C) Pellet fractions were re-suspended in 20 lL of TNE (50 lm Tris/HCl, 150 mm NaCl, 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 on ice After centrifugation, Fc-receptors were blocked by incubation of cells with antibody against CD16 ⁄ CD32 (1 : 100; BD Pharmingen) for 30 on ice After three washes with FACS buffer, primary anti-PrP antibodies (A7 or 12F10) were added in a : 100 dilution in FACS buffer for 45 on ice, washed three times in FACS buffer, and the secondary antibody (FITC-labelled, : 100) was added and incubated for another 45 After the last wash, cells were re-suspended in FACS buffer containing 7-amino-actinomycin 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 stimulation with GpC-ODN-treated cells in an unpaired twotailed t-test using graphpadprism software PrPSc-specific indirect immunofluorescence assay and confocal laser scanning microscopy Cells were plated on glass cover slips (Marienfeld, Germany) at low density They were washed twice in cold NaCl ⁄ Pi and fixed in 4% paraformaldehyde for 30 at 5842 room temperature After sequential treatment with NH4Cl (50 mm in 20 mm glycine), Triton X-100 (0.3%), guanidinium hydrochloride (6 m) and gelatine (0.2%) for 10 each at room temperature and blocking of Fc-receptors, the first antisera were added at : 100 (e.g 4H11) in NaCl ⁄ Pi and incubated for 30 at room temperature After three washes in NaCl ⁄ Pi, FITC- or rhodamine-conjugated secondary 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 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CpG GpC co LPS CpG GpC A 0h co LPS CpG GpC Fig Infection of BV-2 and J774 with 22L prions (A) After stimulation (co, LPS, CpG- ODN, GpC-ODN), BV-2 cells were incubated for 24 h with brain homogenate