Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK1
Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK2
Author for correspondence: Gordon MacPherson. Fax +44 1865 275501. e-mail gordon.macpherson{at}path.ox.ac.uk
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Abstract |
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The consumption of BSE-contaminated meat products is the most likely cause of variant (v) CJD in humans (Bruce et al., 1997 ; Hill et al., 1997
), and ingestion has been implicated in the transmission of other TSE diseases. The timing of events in TSE pathogenesis, as determined by tracking PrPSc accumulation, varies depending on agent strain, host genotype and the route of infection (Farquhar et al., 1994
, 1996
). However, soon after experimental intragastric or oral exposure of rodents with scrapie, infectivity and PrPSc accumulate first in Peyers patches, gut-associated lymphoid tissues and ganglia of the enteric nervous system (Beekes & McBride, 2000
; Kimberlin & Walker, 1989
), long before their detection in the central nervous system (CNS). Likewise, following experimental oral exposure of mule deer fawns with CWD, PrPSc is also detected first in lymphoid tissues draining the gastro-intestinal tract (Sigurdson et al., 1999
). How and when sheep become infected with natural scrapie is not known, but the detection of PrPSc in Peyers patches and gut-associated lymphoid tissues (Andréoletti et al., 2000
; Heggebø et al., 2000
) prior to detection within the CNS (van Keulen et al., 1999
) suggests that this disease is also acquired orally.
Early PrPSc accumulation takes place on follicular dendritic cells (FDCs) within germinal centres in lymphoid tissues of patients with vCJD (Hill et al., 1999 ), sheep with natural scrapie (van Keulen et al., 1996
) and rodents inoculated with scrapie by peripheral routes (Brown et al., 1999
; Mabbott et al., 2000b
; McBride et al., 1992
). In mouse scrapie models, mature FDCs are critical for scrapie replication and PrPSc accumulation in lymphoid tissues, and in their absence neuroinvasion following peripheral challenge is significantly impaired (Brown et al., 1999
; Mabbott et al., 2000a
, b
; Montrasio et al., 2000
).
The transport mechanisms by which TSE agents reach the germinal centres from the gut lumen are not known. Migratory bone marrow-derived dendritic cells (DCs) are centrally involved in transport of proteins both within Peyers patches and on into mesenteric lymph nodes (Banchereau et al., 2000 ). These cells are a distinct lineage from FDCs, which are tissue-resident and are not considered to be of haemopoietic origin (Endres et al., 1999
; Kapasi et al., 1993
). DCs enter the intestinal wall from the bloodstream, sample antigens from the gut lumen, and then migrate via lymph to mesenteric lymph nodes (Liu & MacPherson, 1993
). These observations suggested to us that migrating DCs might provide a cellular bridge between the gut lumen and the secondary lymphoid tissues in which TSE agents replicate.
To test the hypothesis that DCs can acquire TSE agents, we first investigated the uptake of PrPSc by DCs in vitro. Rat bone marrow-derived DCs (BMDCs) were prepared as previously described (Huang et al., 2000 ) and cultured at 1x106 cells/ml in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 40 ng/ml murine GM-CSF and 1500 U/ml rat IL-4. Medium and cytokines were replaced every 72 h, and by day 10 to 12 of culture 90% of cells had characteristic DC morphology and expressed MHC class II, B7 and CD11c. Scrapie-associated fibrils (SAF), highly infective fibrillar aggregates of PrPSc, were prepared from the brains of mice terminally affected with the mouse-passaged ME7 strain as previously described (Hope et al., 1986
), sonicated in PBS, and a suspension equivalent to 10 mg infected brain tissue (wet weight) was added to each BMDC culture for the times indicated. Following incubation, culture medium was aspirated and cells lysed with 0·1% N-laurylsarcosine. Lysates were treated in the presence or absence of 50 µg proteinase K for 30 min at 37 °C, subjected to electrophoresis through 12% SDSpolyacrylamide gels (Bio-Rad) and proteins transferred to polyvinylidine difluoride membranes (Bio-Rad). PrP was detected with rabbit polyclonal antiserum 1B3 specific for PrP (Farquhar et al., 1989
) and bound antibody visualized by enhanced chemiluminescence (Amersham).
Detergent-insoluble, relatively proteinase K-resistant PrPSc accumulations were detected in BMDC lysates within 3 h of culture with SAF, peaking at around 6 h of culture (Fig. 1). No PrPSc accumulations were detected at any time in lysates from BMDCs treated with PBS (Fig. 1
) or SAF equivalent preparation from normal uninfected brain (data not shown). Neither was uptake identified when B or T lymphocytes were incubated with SAF in vitro (data not shown). After antigen acquisition by DCs, a large proportion is degraded in endosomal/lysosomal compartments for presentation to T lymphocytes on MHC class II (Banchereau et al., 2000
). We have shown, however, that DCs, unlike macrophages, can retain some protein antigens in native, non-degraded form for at least 36 h (Wykes et al., 1998
). After 24 h of culture of BMDCs with SAF, the level of PrPSc detected had declined moderately (Fig. 1
), implying that BMDCs acquire PrPSc, some of which is subsequently catabolized but a considerable proportion of which is retained intact. An increased proteinase K-sensitive PrP signal was also detected after 24 h incubation (Fig. 1
), which may also represent the break-up of SAF aggregates within the DC and the revealing of more epitopes as the PrP is digested.
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Immunocytochemical analysis showed that after intra-intestinal SAF exposure, large amounts of PrP were present as conspicuous cytoplasmic inclusions in 4 to 5% of lymph DCs (Fig. 2a). No such deposits were identified within T or B lymphocyte populations (Fig. 2b
, c
, respectively). Much weaker PrP staining was seen around lymph DCs (Fig. 2d
) and B lymphocytes (data not shown) from PBS-treated controls, indicative of membrane-associated endogenously expressed rat PrPc. Immunoblot analysis confirmed the presence of detergent-insoluble, relatively proteinase K-resistant PrPSc in lymph DC lysates selected from SAF-injected rats (Fig. 3
, lane 4). However, the characteristic molecular mass shift in the three-band PrPSc signature after proteinase K digestion was not seen (Fig. 3
, lane 6; Hope et al., 1986
). The multiple bands detected may indicate a difference in PrP processing either in the intestine or within DCs. No PrP was detected in lysates of T or B lymphocytes selected from SAF-injected rats (Fig. 3
, lanes 1, 2, respectively). Neither was PrP detected in lysates from DCs, T or B lymphocytes from mesenteric lymphadenectomized rats treated with PBS or an SAF equivalent preparation from normal uninfected brain as a control (data not shown).
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In this study we show that DCs can acquire PrPSc in vitro and that a small sub-population of migrating DCs can take up and transport PrPSc from the gut lumen through the lymphatics to lymphoid tissue. We have also shown that the uptake of PrPSc from the gut lumen is restricted to DCs, as no PrPSc was detected in other lymph cells or cell-free lymph plasma. The small numbers of cells involved, perhaps in addition to partial intracellular degradative mechanisms, may explain the longer incubation periods, and reduced efficiency of infection, following oral exposure in comparison with other peripheral routes. Within lymphoid tissue FDCs play a critical role in the amplification of TSE infectivity outside the CNS (Brown et al., 1999 ; Mabbott et al., 2000a
, b
; Montrasio et al., 2000
). Our findings suggest that following infection via the gastro-intestinal tract, DCs act as a cellular bridge between the gut lumen and the lymphoid TSE replicative machinery.
Within the intestine, DCs have been described in the lamina propria (Maric et al., 1996 ), and in Peyers patches where they form a dense layer of cells in the subepithelial dome, just beneath the follicle-associated epithelium and in close contact with M cells (Kelsall & Strober, 1996
). Following oral challenge of rodents with scrapie, heavy pathological PrP accumulations are detected within cells of the follicle-associated epithelium with morphology consistent with M cells (Beekes & McBride, 2000
), which have the potential to transcytose infectivity in vitro (Heppner et al., 2001
). However, further studies are necessary to determine whether DCs acquire PrPSc after it has been internalized by M cells, or by direct uptake across the mucosal epithelium as recently shown for the transport of apoptotic intestinal epithelial cells (Huang et al., 2000
) or bacteria (Rescigno et al., 2001
). In addition, our studies do not exclude the possibility of direct uptake into PrP-expressing enteric nerves (Shmakov et al., 2000
).
The detection of infectivity within lymphoid tissues (Bruce et al., 2001 ) and PrP accumulation upon FDCs of patients with vCJD (Hill et al., 1999
) suggest that this disease shares a similar pathogenesis to rodent TSE models. Immunomodulation alters susceptibility to TSEs in rodent models (Mabbott et al., 1998
) and gut inflammation markedly stimulates DC traffic from the intestine (MacPherson et al., 1995
). Our studies suggest that it will be important to investigate where TSEs are taken up in the human gastro-intestinal tract, and whether this can be exacerbated by inflammatory conditions that stimulate DC migration.
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Acknowledgments |
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Footnotes |
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References |
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Received 9 July 2001;
accepted 18 September 2001.