1 Department of Morphology, Genetics and Aquatic Biology, Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway
2 Lasswade Veterinary Laboratory, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
Correspondence
Charles Press
Charles.Press{at}veths.no
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ABSTRACT |
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Published ahead of print on 10 February 2003 as DOI 10.1099/vir.0.18874-0.
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INTRODUCTION |
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The scrapie agent accumulates in lymphoid organs, and the follicular dendritic cell (FDC) in germinal centres has been shown to be the cell of the lymphoid system that sustains replication (Brown et al., 1999; Kitamoto et al., 1991
; McBride et al., 1992
). After natural infection of scrapie in sheep, PrPd has been detected in lymphoid nodules of the Peyer's patches of the gut as early as 5 months after oral infection (Andréoletti et al., 2000
; van Keulen et al., 2000
). The subsequent presence of PrPd in tissues of the enteric nervous system (ENS) in the gut wall has supported the suggestion that the ENS is the site of initial neuroinvasion for the scrapie agent. However, the innervation of lymphoid tissue of the Peyer's patches in sheep is not well documented.
The ENS is a complex system of intrinsic enteric neurons, nerves and supporting cells and extrinsic nerve processes of the sympathetic and parasympathetic nervous systems that are embedded in the wall of the gut and extend from the pharynx to the anal sphincter and into the pancreas and gall bladder (Costa et al., 1987). Within the ENS, two types of nerve meshworks can be distinguished, the ganglionated plexi and the sparse meshworks of aganglionic nerve strands. The major ganglionated plexi are the myenteric (Auerbach's) plexus, located between the circular and longitudinal muscle layers of the gut wall, and the submucosal (Meissner's) plexus (Furness & Costa, 1980
; Timmermans et al., 1992
). In large mammals, including sheep, the submucosal plexus is divided into outer and inner plexi, located close to the inner circular muscle layer and lamina muscularis mucosae, respectively (Balemba et al., 1999
; Timmermans et al., 1992
). However, only limited histochemical and immunocytochemical studies of the innervation of Peyer's patches in ruminants have been undertaken. In cattle, a topographic and structural study of the ENS in jejunum and ileum was performed and demonstrated overall similarities with other species (Balemba et al., 1999
), although there were some differences to the reported organization in the pig (Krammer & Kühnel, 1993
).
Germinal centres are poorly innervated (Felten et al., 1985), and transfer of the scrapie agent from the accumulations of PrPd at these sites to nerve endings of the peripheral nervous system is therefore difficult to explain. Access to peripheral nerves is facilitated if myelination of the nerves is reduced or absent (Kimberlin et al., 1983
). Therefore, the mantle zone of lymphoid follicles that are innervated by terminal unmyelinated nerve fibres has been proposed as the entry point of the scrapie agent into the peripheral nervous system, as this is a region where FDC processes come in close contact with nerve fibres (Glatzel et al., 2000
). The aim of the present study was to investigate the localization of the ENS in the Peyer's patches of sheep and to describe the distribution of PrPd within the ENS of scrapie-affected Suffolk sheep.
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METHODS |
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In the present study, eight sheep (20- to 24-month-old) were studied from the above-mentioned flock which had experienced frequent cases of natural scrapie over a period of several years. At 20 months old, two PrPARQ/ARQ sheep, one PrPARR/ARQ sheep and one PrPARR/ARR sheep were placed in deep anaesthesia and killed by exsanguination. None of these sheep had any clinical evidence of disease. A further four sheep were killed at 23 to 24 months old, having had a short period with clinical signs consistent with scrapie.
From each sheep, tissues from the brain, spinal cord, lymphoid tissues and alimentary tract were collected for histological and immunohistochemical analysis. The results of brain and lymphoid tissue histology and immunohistochemistry have been reported elsewhere (Heggebø et al., 2002). From the alimentary tract of each sheep, the following sites were identified and placed in 10 % neutral-buffered formalin or were quenched in isopentane cooled with liquid nitrogen and subsequently retained in a -70 °C freezer until use: rumen (two sites), omasalreticulum junction, abomasum (pylorus, greater and lesser curvature), duodenum, jejunum (several sites containing grossly identified jejunal Peyer's patches), ileum at the ileocaecal fold and at the ileocolic junction, and colon (adjacent to the ileocaecocolic junction and at the descending colon near to the spiral colon).
Electron microscopy.
The tissues collected from the gut for electron microscopy were immersion-fixed in 2 % periodate-lysine-paraformaldehyde/0·5 % paraformaldehyde for 24 h at 4 °C. Tissues from the gut were cut into 1 mm cubes, post-fixed in 2 % osmium tetroxide, dehydrated and embedded in Araldite. Thick sections were stained by toluidine blue. Mesas were trimmed and 65 nm thick sections were taken from blocks identified as containing representation of the mucosal and Peyer's patches or of the muscularis with ENS ganglia. These sections were placed on 300 mesh nickel grids and post-fixed with 2·5 % glutaraldehyde in PBS. Grids were counterstained with 1 % uranyl acetate and lead citrate.
Immunohistochemistry in paraffin-embedded tissues.
Avidinbiotin complex and peroxidaseanti-peroxidase immunohistochemical staining for disease-associated accumulations of PrP was done using modifications of previously published methods (Haritani et al., 1994). Tissues were subjected to formic acid pre-treatment and hydrated autoclaving. Immunohistochemistry for the detection of PrP was performed using the R521.7 antibody, which was kindly provided by J. Langeveld, IDLO, Lelystad, The Netherlands (van Keulen et al., 1996
), and several other monoclonal and polyclonal anti-PrP sera including P4 and L42 (kindly provided by M. H. Groschup, Germany) (Hardt et al., 2000
) and R486 (kindly provided by R. Jackman, VLA Weybridge, Surrey). The control sections included the use of isotype control sera as the primary antibody.
To enhance the sensitivity of the immunohistochemical method for the detection of PrP in paraffin-embedded tissues, the above procedure was modified to incorporate an additional enhancement procedure, as previously used on frozen tissue (Heggebø et al., 2000). Briefly, following incubation with the peroxidase-conjugated avidinbiotin complex and washing, the sections were incubated with biotinyl tyramide (TSA-Indirect) for 5 min, washed and then incubated with streptavidinhorseradish peroxidase for 30 min. Peroxidase activity was detected using 3-amino-9-ethyl carbazole (Sigma) for 10 min. The reaction was stopped by washing the sections in distilled water. The primary antibody used was L42 and an isotype control serum was used for the control sections. With this enhancement procedure, some weak staining for PrP was detected in the myenteric plexus of formalin-fixed intestines from the PrPARR/ARR sheep and the PrPARR/ARQ sheep. Parallel sections from these resistant sheep that were stained using L42 but without the enhancement steps showed no staining from PrP.
Immunohistochemistry for the detection of cells and nerve fibres of the ENS was performed using polyclonal antibodies against protein gene product 9.5 (PgP 9.5), neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP), all supplied by Dako. For PgP 9.5, an antigen retrieval procedure was used, involving the autoclaving of tissue sections in 0·2 % citrate buffer at 121 °C for 20 min. The primary antibody was incubated overnight at room temperature and immunolabelling was detected using an avidinbiotin complex procedure (ABC kit, Vector Elite) with the substrate chromogen 3,3'-diaminobenzidine and enhancement in 0·5 % aqueous copper sulphate for 3 min.
Histoblot.
For histoblot, frozen sections (12 µm in thickness) were mounted on nitrocellulose (Sigma nitrocellulose membrane, 0·45 µm pore size) and performed in a standard manner (Taraboulos et al., 1992), with some minor modifications (Heggebø et al., 2002
).
Briefly, the tissues were subjected to proteolysis with proteinase K (400 µg ml-1; Serva Electrophoresis) at 55 °C for 4 h. After several pre-treatment steps, the membrane was incubated with the anti-PrP monoclonal antibody L42 overnight at 4 °C. The membrane was incubated with a secondary anti-mouse antibody (Vectastain ABC kit, Vector Laboratories) for 30 min, followed by incubation with streptavidinalkaline phosphatase conjugate (Amersham Pharmacia) for 30 min. A reaction product was produced using BCIP/NBT Pre-mixed Solution (Zymed) for 10 min. The histoblots were examined and images captured using a Leica DC100 digital camera mounted on a Leica MZ 12.5 Stereomicroscope.
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RESULTS |
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Clear differences in innervation of the organized lymphoid tissues were not found between different gut areas, but the Peyer's patches consistently showed, with the exception of the T-cell-dependent areas, a much lower degree of innervation than the gut structures not containing organized lymphoid tissue. No difference in nerve distribution or density was observed when comparing the gut tissues from scrapie-affected and unaffected sheep.
The supportive cells of the ENS, which include Schwann cells, satellite cells and enteric glial cells, were labelled with antibodies to GFAP. Immunolabelling for GFAP was predominantly localized to the ganglia of the myenteric and submucosal plexi but was also detected in the meshwork of nerve fibres interconnecting these plexi within the muscle layers of the gut wall and within the submucosa (Fig. 4b). GFAP-positive fibres were also detected within or closely adjacent to the capsule of the lymphoid nodule (Fig. 4d
).
Disease-specific accumulations of PrP within the ENS
Disease-specific patterns of PrP immunolabelling were found in the enteric neurons, both of the submucosal and myenteric plexi, in all scrapie-affected sheep. However, enteric neuron staining was not found in formalin-fixed intestines of the PrPARR/ARR sheep or of the PrPARR/ARQ sheep. The accumulation of PrP immunolabelling was therefore presumed to be scrapie specific, as previously suggested (van Keulen et al., 1996). Disease-specific accumulations of PrP were detected with each of the antibodies tested and in similar sites in each case. Histoblot examination of the ileal Peyer's patches of scrapie-affected sheep demonstrated the presence of proteinase K-resistant PrP (PrPRES) in locations corresponding to the myenteric plexus, which appeared as a continuous, greyish thin line separate from the strong labelling present in lymphoid nodules (Fig. 5
).
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DISCUSSION |
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In the present study of sheep in the late subclinical and early clinical phase of scrapie, PrPd was consistently present in the ENS when there were also abundant deposits of PrPd in adjacent gut-associated lymphoid tissue. In terminal scrapie in mice (Maignien et al., 1999) and sheep (van Keulen et al., 1999
), PrPd has been detected at sites in the gut extending from the stomach in mice and the rumen in sheep to the colon and rectum. Heggebø et al. (2002)
examined the distribution of PrPd in the lymphoid tissues of the present group of sheep and in the gut found that the dominant localization of PrPd was in the lymphoid nodules of the jejunum, ileum and colon. At other sites such as the abomasum and duodenum, deposits were detected in nodules if these had formed in relation to inflammatory foci. The sparse amounts or absence of PrPd detected in the ENS at these sites was consistent with the previously reported reduced presence of nodular lymphoid tissue (Heggebø et al., 2002
). In a study of natural scrapie in Romanov sheep, Andréoletti et al. (2000)
also found a reduced presence or an absence of PrPd (PrPSc) in the autonomic myenteric nervous plexus at digestive tract sites away from the large lymphoid aggregates of the jejunum and ileum. Drawing support from studies in rodents (Beekes & McBride, 2000
), these investigators suggested that the passage of scrapie from lymphoid structures to the nervous system occurred at the level of nerve fibres innervating these lymphoid organs. However, as noted by Heggebø et al. (2002)
, the general perception that mammalian lymphoid nodules are poorly innervated has cast doubt on the likelihood that neuroinvasion of the scrapie agent occurs within the lymphoid nodule.
A major contribution of the present study was the ultrastructural and immunocytochemical demonstration of a network of nerve fibres within the lymphoid nodules and capsule of the ileal and jejunal Peyer's patches of 20- to 24-month-old Suffolk sheep. This observation indicates that the Peyer's patch nodules provide close contact between nerve fibres and cell populations with abundant PrPd, including FDCs and tingible body macrophages and may represent an important site for neuroinvasion. A link between PrPd-rich FDCs and nerve endings has been sought (Bruce et al., 2000; Glatzel & Aguzzi, 2000
; Glatzel et al., 2001
), but the general observation that mammalian germinal centres are poorly innervated has directed attention to transport of infectivity away from FDCs by dendritic cells and B-cells or the interaction of nerves and FDCs in the mantle zone rather than in the nodule itself. The involvement of noradrenergic neurons in prion neuroinvasion in natural scrapie (Bencsik et al., 2001a
, b
) has been investigated in splenic lymphoid tissue of sheep. These studies showed a close proximity of noradrenergic endings to PrP-expressing cells or PrPSc-accumulating cells but the investigators did not show that tyrosine hydroxylase-positive nerves penetrated lymphoid nodules. The present study used the pan-nerve fibre markers, PgP 9.5 and NSE, to map the distribution of the ENS in association with Peyer's patches and identified fibres within lymphoid nodules. Although a marker such as PgP 9.5 has been reported to label non-neuronal cell populations (Hamzeh et al., 2000
; Langlois et al., 1994
, 1995
), the ultrastructural studies confirmed the presence of nerve fibres within Peyer's patch nodules. Indeed, the relatively high frequency of nerve fibres at the ultrastructural level suggested that, in areas such as the dome and lamina propria, the use of some immunocytochemical markers underestimated the presence of ENS. The presence of nerve fibres in these areas may be of relevance for direct neuroinvasion.
Immunocytochemical studies of the innervation of mammalian lymphoid tissue have mostly been done in rodents and have shown that B-cell-dominated germinal centres contain few or no nerve fibres in contrast to the rich network in the surrounding T-cell regions (Felten et al., 1985; Felten & Felten, 1988
; Lorton et al., 1991
). The present study differs from these earlier neuroanatomical works in many respects, including the species and tissue examined, and the age and disease status of the investigated animals. Few studies have addressed the distribution of nerves and supporting cells in the ENS of Peyer's patches of sheep but studies performed in cattle (Balemba et al., 1999
) and pigs (Krammer & Kühnel, 1993
) did not report the presence of nerve fibres in nodules, although other studies in cats (Fehér et al., 1992
; Ichikawa et al., 1994
), pigs (Kulkarni-Narla et al., 1999
) and hamsters (Pfoch & Unsicker, 1972
) have reported nerve fibres in nodules. A feature of the biology of gut-associated lymphoid tissue in cattle, pigs and sheep is that the large continuous aggregate of lymphoid tissue in the ileum and distal jejunum undergoes involution around the time of sexual maturity (Griebel & Hein, 1996
; Reynolds & Morris, 1983
). While the studies in cattle and pigs did not consider the influence of involution on innervation, studies in rodents and birds have shown that the innervation of lymphoid organs changes with age (Felten et al., 1987
). With involution, tissues such as the thymus (Madden et al., 1998
) and bursa of Fabricius (Ciriaco et al., 1995
) experience an apparent increase in the density of noradrenergic fibres that may be related to altered immune function. Whether changes occur in the density of nerve fibres in the lymphoid nodules of ageing sheep needs to be investigated. Furthermore, the disease status of the animals in the present study may have influenced the distribution of the ENS, although nerve fibres were found in lymphoid nodules of Peyer's patches from the gut of both scrapie-affected and -unaffected sheep. While this observation would suggest that the presence of nerve fibres is not a consequence of scrapie infection, the possibility that scrapie infection and the accumulation of PrPd in lymphoid nodules influences innervation also needs to be studied further. A preliminary study of young sheep (1- to 2-month-old) did not find nerve fibres in gut-associated lymphoid nodules (L. González, unpublished observation).
The patterns of immunolabelling for PrPd resembling fibre-like structures were not obtained in nodules and have not been reported by others investigating the accumulation of PrPd in the gut-associated lymphoid tissue of sheep (Andréoletti et al., 2000; Heggebø et al., 2002
; van Keulen et al., 1999
; Jeffrey et al., 2001a
). The use of enhancement protocols in the present study to increase the sensitivity of the immunohistochemical procedure did not result in the identification of PrPd profiles consistent with nerve fibres. It is proposed that spread of PrPd along peripheral nerves occurs by established axonal transport mechanisms and that in nerve fibres the scrapie agent is in transit rather than being actively replicated (McBride et al., 2001
), which may preclude immunohistochemical detection. Immuno-electron microscopy studies will be important in resolving the role of nervous tissue within gut-associated lymphoid nodules for neuroinvasion in sheep.
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ACKNOWLEDGEMENTS |
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Received 3 October 2002;
accepted 27 January 2003.