Journal of Histochemistry and Cytochemistry, Vol. 50, 1357-1370, October 2002, Copyright © 2002, The Histochemical Society, Inc.


ARTICLE

Phenotyping of Protein–Prion (PrPsc)-accumulating Cells in Lymphoid and Neural Tissues of Naturally Scrapie-affected Sheep by Double-labeling Immunohistochemistry

Olivier Andréolettia, Patricia Berthonb, Etienne Levavasseurb, Daniel Marcb, Frédéric Lantierb, Eoin Monksc, Jean-Michel Elsend, and François Schelchera
a UMR INRA-ENVT, Physiopathologie Infectieuse et Parasitaire des Ruminants, Ecole Nationale Vétérinaire, Toulouse, France
b INRA, Laboratoire de Pathologie Infectieuse et Immunologie, Nouzilly, France
c Department of Agriculture, Food and Rural Development, Central Veterinary Research Laboratory, Abbotstown, Castleknock, Dublin, Ireland
d INRA, Station d'Amélioration Génétique des Animaux, Auzeville, France

Correspondence to: Olivier Andréoletti, Physiopathologie Infectieuse et Parasitaire des Ruminants, Ecole Nationale Vétérinaire, 23 Chemin des Capelles, 31076 Toulouse Cedex 3, France. E-mail: o.andreoletti@envt.fr


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by amyloid deposition of protein–prion (PrPsc), the pathogenic isoform of the host cellular protein PrPc, in the immune and central nervous systems. In the absence of definitive data on the nature of the infectious agent, PrPsc immunohistochemistry (IHC) constitutes one of the main methodologies for pathogenesis studies of these diseases. In situ PrPsc immunolabeling requires formalin fixation and paraffin embedding of tissues, followed by post-embedding antigen retrieval steps such as formic acid and hydrated autoclaving treatments. These procedures result in poor cellular antigen preservation, precluding the phenotyping of cells involved in scrapie pathogenesis. Until now, PrPsc-positive cell phenotyping relied mainly on morphological criteria. To identify these cells under the PrPsc IHC conditions, a new, rapid, and highly sensitive PrPsc double-labeling technique was developed, using a panel of screened antibodies that allow specific labeling of most of the cell subsets and structures using paraffin-embedded lymphoid and neural tissues from sheep, leading to an accurate identification of ovine PrPsc-accumulating cells. This technique constitutes a useful tool for IHC investigation of scrapie pathogenesis and may be applicable to the study of other ovine infectious diseases.

(J Histochem Cytochem 50:1357–1370, 2002)

Key Words: PrPsc double labeling, immunohistochemistry, ovine scrapie


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The pathogenesis of transmissible spongiform encephalopathies (TSEs), such as scrapie in sheep (Hadlow et al. 1982 ; van Keulen et al. 1996 ) and in rodent models (McBride et al. 1992 ; Maignien et al. 1999 ), as well as the variant form of Creutzfeldt–Jakob disease in humans (Hill et al. 1999 ), usually involves a primary replication in the immune system followed by a spread to the central nervous system (CNS).

In sheep, the dissemination pathway of the prion agent appears to depend on the genetic susceptibility of the host to the disease. Genetic resistance of sheep to scrapie is mainly controlled by polymorphisms at codons 136, 154, and 171 of the PrP gene. In agreement with data from various European sheep flocks with natural scrapie (Bossers et al. 1996 ; Hunter et al. 1996 ), a recent study of a Romanov flock affected by natural scrapie (Elsen et al. 1999 ) confirmed that V136R154Q171/VRQ, VRQ/ARQ, and ARQ/ARQ were the most susceptible genotypes. Animals with the VRQ/ARR genotype showed moderate disease susceptibility, whereas ARR/ARR sheep were highly resistant, with no scrapie occurring within this group.

Replication of the agent in lymphoid tissues is not an absolute requirement for development of the disease, because it is not observed in scrapie-affected VRQ/ARR sheep (van Keulen et al. 1996 ; Schreuder et al. 1998 ; Andreoletti et al. 2000 ). However, neuro-invasion appears to be much more efficient when this primary replication step occurs, as seen in highly susceptible VRQ/VRQ, VRQ/ARQ, and ARQ/ARQ sheep (Andreoletti et al. 2000 ).

Neurodegeneration in TSEs is characterized by vacuolar changes of the neuropil and neurons in the gray matter, gliosis, and neuronal loss (Masters and Richardson 1978 ), and by accumulation of an abnormal isoform (PrPsc) of the prion protein in neural tissues and, in specific instances, lymphoid tissues. The normal cellular isoform of the prion–protein (PrPc), is encoded by the PrP gene (Oesch et al. 1985 ) and is constitutively expressed by many cell types (Bendheim et al. 1992 ; Horiuchi et al. 1995 ). Although the true nature of the TSE causative agent is still unknown, expression of this host cellular protein is a prerequisite to development of the disease (Bueler et al. 1993 ). At present, the only biochemical marker specific for TSE is PrPsc, the pathogenic isoform of normal cellular PrPc. PrPsc is distinguishable from PrPc by its greater resistance to proteolysis and to high temperatures (Harris 1999 ).

The disease-specific PrPsc accumulations are mainly composed of deposits in the B-follicle germinal centers of lymphoid formations (McBride et al. 1992 ; van Keulen et al. 1996 ; Andreoletti et al. 2000 ) and in the neuropil, around blood vessels and neurons, and within neurons at the CNS level (van Keulen et al. 1995 ; Jeffrey et al. 1998 ).

Many studies on the pathogenesis of experimental and natural scrapie have been carried out with a PrPsc IHC approach to phenotype cells involved in the infectious process and to determine the dissemination pathways within the host (van Keulen et al. 1995 , van Keulen et al. 1996 ; Jeffrey et al. 1998 , Jeffrey et al. 2001 ; Andreoletti et al. 2000 ; Foster et al. 2001 ; Sigurdson et al. 2001 ). Most anti-PrP antibodies recognize both isoforms of the protein. In addition, the amyloid deposition of PrPsc induces buried epitopes. Therefore, specific and sensitive in situ detection of the PrPsc requires both suppression of PrPc antigenicity and a PrPsc antigen retrieval step to enhance amyloid antigenicity (Hayward et al. 1994 ; van Everbroeck et al. 1999 ). The most efficient pretreatments used to enhance the specific immunolabeling of PrPsc consist of incubation of tissue sections in formic acid (Kitamoto et al. 1987 ) followed by a hydrated autoclaving step at 121C (Haritani et al. 1994 ). Such chemical and heat pretreatments require the use of formalin-fixed, paraffin-embedded tissue sections. These PrPsc-specific IHC procedures, which are standardized for prion disease diagnosis (Bell et al. 1997 ), are not optimal for the other cell antigens, such as cluster of differentiation (CD) antigens, and hamper the setting up of PrPsc double-labeling protocols with homologous anti-CD antibodies.

In studies of scrapie pathogenesis, identification of PrPsc-positive cells relied on their morphology and tissue localization. These mainly concerned astrocytes and neurons in the CNS (van Keulen et al. 1995 ; Foster et al. 1996 ) as well as follicular dendritic cells (FDCs) and macrophages in the immune system (van Keulen et al. 1996 ; Brown et al. 1999 ; Beekes and McBride 2000 ). Until now, double-labeling techniques clearly showed localization of PrPsc in astrocytes (van Keulen et al. 1995 ) and in microglial cells (Miyazono et al. 1991 ; Guiroy et al. 1994 ) at the CNS level, with no data available on cells involved in the immune system.

Our objective was to better understand scrapie pathogenesis in sheep, and particularly to determine the cell populations in the central nervous and immune systems in which PrPsc accumulated. These cells might constitute either support of prion replication or the main targets of scrapie pathology. The purpose of this work was to identify the cell phenotype of PrPsc-positive cells as well as the cell structures involved in PrPsc deposition in lymphoid and neural tissues of clinically scrapie-affected sheep. Therefore, we attempted to develop a simple and sensitive PrPsc double-labeling technique that might be applied to the identification of ovine cell populations and structures involved in PrPsc accumulation in both lymphoid and central neural tissues. For this purpose, we screened a panel of heterologous antibodies that could be used under standard PrPsc IHC conditions and that crossreacted with ovine cellular antigens of both lymphoid and CNS tissues. These antibodies were selected according to their known reactivity to formalin-resistant epitopes of human cellular antigens and to protein markers highly conserved among mammalian species. In the lymphoid tissues, we searched for specificity to T-lymphocytes, B-lymphocytes, FDCs, macrophages, and proliferating cells. In the CNS tissues, we looked for antibodies that distinguished astrocytes, microglial cells, neurons, neuronal processes, and synapses.


  Materials and Methods
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Materials and Methods
Results
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Literature Cited

Animals and Samples
Fifteen clinically scrapie-affected and 10 healthy sheep, from three different flocks and two breeds (Romanov and Manech Redface), were used for this study. Their PrP genotypes at codons 136, 154, and 171 were determined from blood samples (Labogena; Jouy-en-Josas, France). The scrapie-affected group, expressing clinical signs of scrapie, was composed of 11 genetically susceptible (five VRQ/VRQ and six ARQ/ARQ) and four intermediate (VRQ/ARR) animals. The scrapie diagnosis was confirmed by histological examination (neuronal and neuropil vacuolation in the gray matter, gliosis) of the brainstem (obex). The healthy control group consisted of five genetically resistant (ARR/ARR), two intermediate (ARQ/ARR), and three susceptible (ARQ/ARQ) sheep with no detectable histopathological lesions. Animals were raised according to the requirements of the INRA Animal Care and Ethics Committee. All procedures on the animals were performed by workers accredited by the French Ministry of Agriculture and were aimed at limiting animal pain and distress.

Sheep were sacrificed by an IV injection of pentobarbital (10 mg/kg) followed by exsanguination. Caudal brainstem (obex), palatine tonsils, mesenteric lymph nodes, and spleen were rapidly removed and fixed in neutral-buffered 10% formalin (4% formaldehyde) for 4–10 days before paraffin embedding, according to our standard IHC procedures for scrapie diagnosis. Tissue sections 2 µm thick were collected onto adhesive-treated slides (ChemMate Capillary Gap Microscope Slides, S 2024; DAKO, Trappes, France) and dried overnight at 56C before being deparaffinized and rehydrated.

Immunohistochemical Methods
Anti-PrP Antibodies. PrPsc immunolabeling was carried out using two anti-PrP antibodies raised against ovine PrP peptides: either the rabbit polyclonal antibody (PAb) R-521 (ovine PrP 94-105; kindly provided by Dr. L.J.M. van Keulen, ID-Lelystad, The Netherlands; Garssen et al. 2000 ) or the mouse monoclonal antibody (MAb) 2G11 (ovine PrP 146-R154-R171-182 and specifically recognizing the R151-R159 sequence), as previously described (Andreoletti et al. 2000 ). Under our IHC conditions, MAb 2G11 appeared to be highly specific for the ovine PrPsc, generating no labeling of the PrPc in tissues of sheep from a scrapie-free flock (Andreoletti et al. 2000 ). These two anti-PrP antibodies were both able to detect PrPsc deposition in organs of scrapie-affected sheep, whatever their PrP genotype (data not shown) and were routinely used for scrapie diagnosis.

Antibodies Used for Ovine Lymphoid Cell Phenotyping. Because the majority of MAbs raised against ovine CD antigens react poorly with their cognate ligands in formalin-fixed and paraffin-embedded sheep tissue sections, the crossreactivity of anti-human CD antibodies with processing-resistant epitopes of ovine CD antigens was assessed. A panel of anti-CD antibodies, selected for their immunoreactivity to either formalin-resistant epitopes or highly conserved mammalian cellular proteins, was screened for the identification of ovine T-cells, immature and mature B-cells, FDCS, macrophages, and proliferating cells. Details of all antibodies tested are presented in Table 1.


 
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Table 1. Screening results of antibodies tested for cell phenotyping using ovine paraffin-embedded lymphoid tissue sectionsa,b

Antibodies Used for Ovine CNS Cell Identification. Different markers were investigated to identify cells and structures involved in PrPsc accumulation. A rabbit polyclonal serum raised against bovine glial fibrillary acidic protein (GFAP) was used to label astrocytes. A rabbit PAb specific for the bovine S100 protein was used to identify neuroectoderm-derived cell populations (neurons and astrocytes). MAbs specific for bovine synaptophysin and human neurofilament protein (70 and 200 kD) were used to investigate the PrPsc distribution in neuronal synapses and processes, respectively. Finally, according to the labeling results of the monocyte/macrophage lineage on lymphoid tissues (see Results), the anti-human CD68 MAb Ki-M6 was also used to immunostain microglial cells (Ulvestad et al. 1994 ) (Table 2).


 
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Table 2. Screening results of antibodies tested for cell phenotyping and cell structure identification using paraffin-embedded tissue sections from the ovine CNSa

Antigen Retrieval Protocols
Our antigen retrieval protocol comprised a mild proteolysis followed by hydrated buffered autoclaving (McNicol and Richmond 1998 ). Antibody screening was first performed using autoclaving buffers of various pH. After rehydration, tissue sections were first subjected to a 5-min proteolysis in a 0.1 M Tris-buffered saline solution (TBS), pH 7.6, containing 0.1% trypsin (200 FIP-U/g; EC 3.4.21.4; Merck, Gradignan, France) at 37C. They were then autoclaved in a pressure cooker for 5 min at 121C in (a) 10 mM citrate buffer (pH 3.0), (b) 10 mM citrate buffer (pH 6.1), or (c) 1 mM EDTA solution (pH 8.0) and allowed to cool for 20 min.

PrPsc-specific labeling requires an initial formic acid treatment before the antigen retrieval steps (Kitamoto et al. 1987 ). Therefore, all antibodies selected for PrPsc double labeling were also tested under this protocol. Our usual protocol for PrPsc labeling involved a 30-min incubation at room temperature (RT) in 98% formic acid (Merck) followed by a retrieval protocol that included the proteolysis step and autoclaving in 10 mM citrate buffer (pH 6.1) for 5 min (Andreoletti et al. 2000 ).

Single-labeling Method
Endogenous peroxidase was inhibited using a 1:100 dilution of hydrogen peroxide 30% (w/w) in methyl alcohol for 30 min at RT. Sections were then washed with tapwater. Nonspecific binding sites were blocked by incubating sections with 20% normal goat serum in TBS for 20 min. Primary antibody was then applied at the chosen dilution (see Table 1 and Table 2) for 60 min at RT. Then a 30-min incubation with a biotinylated secondary goat antibody (1:100 diluted), specific for either rabbit or mouse immunoglobulins (Ig), was performed before application of a streptavidin–peroxidase complex (1:100 diluted) for 30 min. Revelation was performed using 3,3'-diaminobenzidine (DAB) (ChemMate Detection Kit Peroxidase/DAB, K 5001; DAKO). Each step was followed by three 5-min washes in TBS containing 1% skimmed milk and 0.05% Tween-20. Sections were counterstained with Mayer's hematoxylin.

Double-labeling Method
We developed a novel two-step method based on the simultaneous application of the two primary antibodies, each raised in a different species, followed by a simultaneous incubation with two secondary antibodies, each appropriate to the species of origin of the primary and each carrying a separate enzyme label. Typically, couples of primary antibodies consisted of a mouse MAb and a rabbit PAb. When the CD antigen was revealed using a PAb, the PrPsc was detected with an MAb and vice versa. Each antibody was used at its optimal dilution as determined by single-labeling protocols. Antibodies were diluted in TBS containing 1% bovine serum albumin (BSA, albumin fraction V; Merck). The first secondary antibody chosen to reveal the CD antigen was a goat antibody specific for either mouse or rabbit Ig. This was directly coupled to a dextran polymer carrying peroxidase (EnVision, K 4001 or K 4003; DAKO) and was supplemented with 5% normal sheep serum. The other secondary antibody revealing the anti-PrPsc antibody was a classical biotin-labeled goat antibody specific for either rabbit Ig (1:200 diluted) (E 0432; DAKO) or mouse Ig (1:300 diluted) (E 0433; DAKO), depending on the nature of the anti-PrP antibody used. The mixed secondary antibodies were applied for 30 min at RT. An alkaline phosphatase–streptavidin complex (1:100 diluted) (P 0397; DAKO) was then used for 30 min at RT to amplify the PrPsc-specific signal.

Revelation was performed sequentially using first the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT, dark blue-purple endproduct; K 0598; DAKO) for 5–10 min and then the peroxidase chromogen using either 3-amino-9-ethylcarbazole (AEC+, red endproduct; K 3461; DAKO) or DAB (brown endproduct; K 3465; DAKO) for 3–20 min. Sections were rinsed in tapwater between these two final steps. They were counterstained with Mayer's hematoxylin.

Controls
To characterize nonspecific immunolabeling, each IHC run included negative serum controls in which the primary antibody was either omitted or replaced by normal rabbit or mouse serum. In addition, mouse MAbs were replaced by isotype-matched MAbs irrelevant to the investigated tissue. Reproducibility of the immunostaining was assessed using serial tissue sections from the same sample included in two successive IHC runs. For double labeling, crossreactivity controls were performed for each couple of primary antibodies and each sample to verify the absence of inter-species reactivity of secondary antibodies towards primary antibodies. The absence of a possible affinity between the two secondary antibodies was also checked.


  Results
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Materials and Methods
Results
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Literature Cited

PrPsc Immunolabeling
The MAb 2G11 and PAb R521 gave similar results, as previously described (Andreoletti et al. 2000 ). No nonspecific labeling was observed in negative control sections and no PrPsc was observed in tissues from the 10 healthy sheep, whatever their PrP genotype (ARR/ARR, ARQ/ARR, or ARQ/ARQ). From the four VRQ/ARR animals of the group expressing clinical signs of scrapie, PrPsc was detected in the CNS but not in lymphoid tissues. PrPsc deposits were observed in both lymphoid and neural tissues from the 11 susceptible VRQ/VRQ and ARQ/ARQ sheep at the clinical stage of the disease.

Screening of the Anti-CD Antibodies
Results of the screening of antibodies tested for cell phenotyping on ovine paraffin-embedded lymphoid tissue sections are presented in Table 1.

Ovine T-lymphocytes were labeled with anti-CD3 antibodies only. A majority of cortical interfollicular cells and a few centrofollicular cells were labeled. The immunostaining was membranous, intense, non-granular, and sometimes associated with light and diffuse cytoplasmic labeling. Optimal results were obtained with PAb A 0452. All the pretreatments tested, including the PrPsc-specific protocol, allowed clear labeling of sheep T-cells (Fig 1A).



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Figure 1. Mesentric lymph nodes from VRQ/VRQ sheep with natural scrapie. (A,B) Double labeling of PrPsc deposition (Mab 2G11, alkaline phosphatase, BCIP/NBT) and of the CD3-positive T-lymphocytes (Pab A0452, peroxidase, AEC). (A) Distribution of CD3-positive cells in a B-follicle using PAb A0452 following the PrPsc labeling protocol. Bar = 100 µm. (B) PrPsc deposits were not observed in the cytoplasm of CD3-positive cells. Bar = 12.5 µm. (C,D) Double labeling of PrPsc deposition (PAb R521, alkaline phosphatase, BCIP/NBT) and of the CD20-like positive B-lymphocytes (MAb BLA36, peroxidase, AEC). (C) Labeling pattern obtained with MAb BLA36 in a B-follicle under the PrPsc labeling protocol. Bar = 100 µm. (D) No PrPsc deposition was observed in the cytoplasm of CD20-positive cells. Bar = 12.5 µm. (E,F) Double labeling of PrPsc deposition (PAb R521, alkaline phosphatase, BCIP/NBT) and of the CD79{alpha}cy-positive B-lymphocytes (MAb HM57, peroxidase, DAB), without nuclear counterstaining to better identify lightly labeled CD79{alpha}cy-positive cells. (E) Mab HM57 immunostaining of CD79{alpha}cy-positive cells in a B-follicle following the PrPsc labeling protocol. Bar = 50 µm. (F) No PrPsc deposition was detected in the cytoplasm of CD79{alpha}cy-positive cells. Bar = 12.5 µm.

Among the antibodies tested to identify ovine B-lymphocytes, only two, MAb BLA36 (anti-CD20-like) (Fig 1C) and MAb HM57 (anti-CD79{alpha}cy) (Fig 1E), allowed specific labeling of these cells. Using the anti-CD79{alpha}cy MAb HM57, only a few lymphocytes, mainly located in the dark zone of B-follicles, were positive. On morphological criteria, the most immature cells showed the strongest labeling. Mature B-cells were poorly or not at all labeled. Labeling was both cytoplasmic and membranous, non-granular, and of moderate intensity. Hydrated autoclaving in EDTA, pH 8.0, or citrate, pH 6.1, produced better results than the PrPsc-specific pretreatment, in which the formic acid incubation slightly decreased the signal. Optimal results with the anti-CD20-like MAb BLA36 were obtained after a pretreatment with EDTA, pH 8.0, while the PrPsc antigen retrieval led to a slight decrease of the signal. Morphologically immature B-lymphocytes in the dark zone of B-follicles were either weakly positive or negative. Conversely, more mature B-cells were strongly positive, although plasmocytes appeared unlabeled. The signal was membranous, non-granular, and strong, and was sometimes associated with light diffuse cytoplasmic labeling.

Concerning the ovine FDC, both anti-CD21 and anti-CD35 MAb remained negative on paraffin sections. Only the CNA.42 MAb was able to label this cell subset (Fig 2A). The signal was intense using autoclaving in either citrate, pH 6.1, or EDTA, pH 8.0, and PrPsc-specific pretreatment also allowed a quite distinct labeling. The FDC network was revealed by fine intracytoplasmic granules, which formed more compact clusters in the vicinity of the nucleus. Specific labeling of the vascular endothelium, such as that described in humans (Raymond et al. 1997 ), was also observed.



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Figure 2. Lymphoid tissues from VRQ/VRQ sheep with natural scrapie. (A) Single immunolabeling of the follicular dendritic cell network in a B-follicle germinal center of the mesenteric lymph node (MAb CAN.42, peroxidase, DAB. Bar = 75 µm. (B) PrPsc deposition (PAb R521, alkaline phosphatase, BCIP/NBT) in follicular dendritic cell cytoplasm (MAb CAN.42, peroxidase, AEC). B = 30 µm. (C) PrPsc granule (PAb R521, alkaline phosphatase, BCIP/NBT) in a macrophage-like, CD68-positive cell (MAb Ki-M6, peroxidase, AEC). Bar = 10 µm. This double-labeled CD68-positive cell contained a phagocytosed cell nucleus (blue). (D) Double labeled of PrPsc deposition (PAb R521, alkaline phosphatase, BCIP/NBT) and of the B-follicle proliferating cells expressing the nuclear Ki-67 antigen (MAb MIB-1, peroxidase, AEC), with no nuclear counterstaining. Bar = 20 µm. No PrPsc deposition was identified in the cytoplasm of such proliferating cells.

Most of the antibodies tested against ovine macrophages produced negative results with paraffin sections. The anti-ovine CD14 antibody (VPM 65) induced only weak labeling after citrate, pH 6.1, and EDTA, pH 8.0, which was considered not significant enough for our purpose. The formic acid treatment inhibited the binding of this MAb to the ovine CD14 and did not allow its use in PrPsc double labeling. The anti-lysozyme antibody (PAb A 0099) generated labeling of only a limited macrophage subset. The signal was cytoplasmic, granular, and intense. Optimal results were obtained using either citrate, pH 6.1, or EDTA, pH 8.0, while the PrPsc-specific pretreatment resulted in a strong decrease of the labeling. One anti-CD68 MAb (Ki-M6) provided excellent results whatever the pretreatment. The staining was even increased after formic acid treatment, as previously described (Muhleisen et al. 1995 ). Labeling was cytoplasmic, granular, and very strong (Fig 2C). Positive cells had morphological features of macrophages and interdigitating dendritic cells.

Assays performed on ovine proliferating cells using the MAb MIB-1 specific for the nuclear Ki-67 antigen gave excellent results regardless of the pretreatment used. Nuclei of proliferating cells were strongly positive (Fig 2D). No cytoplasmic deposits were observed. Positive cells were located mainly in the B-follicle germinal centers, and a minority of them were distributed in the interfollicular area.

This screening led to the selection of a panel of anti-CD antibodies that could be used under our PrPsc IHC protocol to allow simultaneous identification of ovine T- and B-lymphocytes, FDCs, macrophages, and proliferating cells in paraffin-embedded lymphoid tissues.

Screening of Antibodies Specific for Ovine CNS Markers
All antibodies screened were able to induce specific labeling whatever the pretreatment tested (Table 2). The MAb 2F11 signal on neurofilaments was partially decreased after formic acid treatment, but the labeling, which was initially non-granular and intense, remained strong enough for a PrPsc double-labeling assay. Anti-GFAP antibody (PAb Z 0334) gave intense cytoplasmic labeling in astrocytes, whereas anti-S100 protein antibody (PAb Z 0311) induced a similar labeling in both astrocytes and neurons. MAb Ki-M6, specific for the human CD68 antigen, generated in the CNS tissues a light, granular, and cytoplasmic labeling of the ovine microglial cells, similar to that observed with the macrophage lineage in lymphoid tissues. They appeared as small ovoid cells with many long and thin ramifications, and were concentrated mainly in perivascular and perineuronal positions. Anti-synaptophysin MAb SY 38 induced fine granular but intense deposits in neuronal perikarya, and also in neuronal processes, allowing the identification of synaptic areas.

PrPsc Double Labeling in Ovine Lymphoid Tissues and CNS
Couples of primary antibodies used in this work and their optimal dilutions are presented in Table 3.


 
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Table 3. Technical parameters for pairs of antibodies used in PrPsc double-labeling protocolsa

In lymphoid tissues of clinically scrapie-affected VRQ/VRQ and ARQ/ARQ sheep, PrPsc deposits were localized in many but not all CD68-positive cells (Fig 2C). Clear PrPsc deposits in FDC cytoplasmic processes were also observed in these animals (Fig 2B). None of the lymphocytes, either T-lymphocytes (CD3-positive cells), mature B-cells (CD20 like-positive cells), or immature B-cells (CD79{alpha}cy-positive cells), appeared to contain PrPsc granules in their cytoplasm (Fig 1B, Fig 1D, and Fig 1F). Only a few B- or T-lymphocytes showed PrPsc granules on the outer surface of their plasma membranes. None of the proliferating Ki-67-positive cells showed cytoplasmic PrPsc deposits, suggesting that PrPsc-positive cells were post-mitotic (Fig 2D).

In the caudal brainstem (obex) of clinically scrapie-affected sheep, whatever their PrP genotype, the PrPsc labeling pattern suggested its accumulation in neuronal perikarya and processes (Fig 3A) and in glial cells (Fig 3B). Double labeling confirmed the localization of PrPsc deposits in neuroectodermic cells (S100 protein-positive cells) (Fig 3C) and astrocytes (GFAP-positive cells) (Fig 3D). PrPsc granules were observed in cytoplasmic processes and just outside their outer membrane. Using this technique, we were unable to determine whether this PrPsc was free or bound to cells (neurons or glial cells). PrPsc deposits were also present in a few microglial cells, mainly as large perinuclear granules (data not shown).



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Figure 3. Immunohistochemical identifications of PrPsc-positive cells in CNS tissue (obex area) from VRQ/VRQ sheep with natural scrapie. (A,B) PrPsc single immunolabeling in the brainstem (MAb 2G11, peroxidase, DAB) in neurons and glial cells, respectively. Bars = 10 µm. PrPsc deposits in neurons were located in both perikarya and neuronal processes. Some granules were close to the neuronal cell membrane. (C) Double immunostaining of PrPsc accumulation (MAb 2G11, alkaline phosphatase, BCIP/NBT) and of neuroectodermic cells (neurons and astrocytes), using an anti-S100 protein antibody (PAb Z 0311, peroxidase, AEC). Bar = 5 µm. (D) PrPsc accumulation (MAb 2G11, alkaline phosphatase, BCIP/NBT) in GFAP-positive astrocytes (PAb Z 0334, peroxidase, AEC). Bar = 5 µm. (E) PrPsc (PAb R521, alkaline phosphatase, BCIP/NBT) deposits could be identified in distal neuronal processes using an anti-neurofilament antibody (MAb 2F11, peroxidase, AEC). Bar = 5 µm. (F) Some PrPsc deposits (PAb R521, alkaline phosphatase, BCIP/NBT) could be observed at the level of synaptic areas (MAb SY 38, peroxidase, DAB). Bar = 2 µm.

The accumulation of PrPsc in neuronal perikarya was obvious. Its deposition was also observed in cell structures expressing neurofilament proteins of 70 and 200 kD, pointing to an intra-axonal and intra-dendritic distribution of PrPsc (Fig 3E). PrPsc deposits lining neurofilament-positive processes were also observed, suggesting a possible deposition on neuronal outer membranes. Many structures that were positive for both synaptophysin and PrPsc were identified in neuronal processes, strongly suggesting PrPsc deposition at the synaptic level (Fig 3F).


  Discussion
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The aim of this work was to set up a PrPsc double-labeling protocol leading to the identification of cells involved in scrapie pathogenesis, whatever the stage of the disease, to precisely define the dissemination pathways of the prion agent within its host.

Standard IHC protocols established for prion disease diagnosis are based on pretreatments of tissue sections that denature PrPc and increase PrPsc antigenicity. These treatments, usually involving formic acid incubation and hydrated autoclaving, require the use of formalin-fixed, paraffin-embedded tissue sections (van Keulen et al. 1995 ; Bell et al. 1997 ) and could generate major alterations in tissue morphology. In a previous unpublished study using anti-PrP MAb 2G11, we set up a protocol that improved the sensitivity of PrPsc labeling and avoided significant alteration of tissue morphology. Studies on scrapie pathogenesis require very sensitive assays and, under our IHC conditions, a formic acid incubation for 30 min followed by a hydrated autoclaving step is necessary for precise detection of PrPsc granules (van Keulen et al. 1995 , van Keulen et al. 1996 ; Andreoletti et al. 2000 ). Our method has been evaluated in procedures for the standardization of IHC protocols for PrPsc detection in ovine palatine tonsils (European proposal FAIR-CT 98-6013) and in ovine and bovine brainstems (European proposal FAIR-CT 98-7021), and has been already selected as reference protocol in the European proposal FAIR-CT 98-6013.

Recent reports have described new methodologies applied to prion–protein IHC. Ovine PrPsc was specifically detected in frozen lymphoid tissue sections (Heggebo et al. 2000 ). In the absence of pretreatments denaturing the PrPc antigenicity, the lack of PrPc detection after this protocol may result from the low PrPc expression in lymphoid organs compared to the CNS (Moudjou et al. 2001 ). On the other hand, fixation procedures before paraffin embedding have been reported recently, allowing either a homologous detection of the CD antigens with the use of a non-aldehyde fixative containing zinc salts (Gonzalez et al. 2001 ) or an improvement of the PrPsc IHC with Carnoy's fixative (Giaccone et al. 2000 ). However, these two procedures do not appear to be compatible with the formic acid treatment that is used to reduce scrapie infectivity of tissue sections (Taylor et al. 1997 ) and also to enhance the amyloid antigenicity of PrPsc (Kitamoto et al. 1987 ).

Although studies on natural scrapie in sheep have shown a great variation in neuropathological lesions, the brainstem, particularly its caudal part, the medulla oblongata (obex), appears to be consistently involved with notably the presence of PrPsc deposits in the dorsal motor nucleus of the vagus nerve (van Keulen et al. 1995 ; Wood et al. 1997 ; Ryder et al. 2001 ; Sigurdson et al. 2001 ). Therefore, in this study the presence of the prion agent within the CNS was assessed by histological and IHC examinations of the obex. After our protocol, PrPsc was detected in all the 15 sheep (five VRQ/VRQ, six ARQ/ARQ, and four VRQ/ARR) expressing clinical signs of scrapie, in correlation with the histopathological changes observed in the obex. The homozygous VRQ and ARQ sheep are considered the most susceptible to scrapie, expressing clinical signs of scrapie before 24 months of age but with different disease incidences (80% and 45%, respectively). VRQ/ARR and ARQ/ARR sheep are characterized by a lower scrapie susceptibility (less than 5% of the group affected) with a longer incubation period of 5–7 years (Elsen et al. 1999 ). PrPsc was present in large amounts in lymphoid organs and brains of susceptible sheep (VRQ/VRQ and ARQ/ARQ) from this scrapie-affected group. More interesting is the finding of PrPsc deposits in brains, but not in lymphoid organs, of clinically scrapie-affected VRQ/ARR animals. This feature appears characteristic of scrapie pathogenesis in heterozygous ARR sheep, because it was observed in all the eight such animals that we examined in the present and previous studies. Although this observation is based on a few cases only, as a result of the very low scrapie incidence in exposed animals from this PrP genotype (Elsen et al. 1999 ), it agrees well with similar findings published by others (van Keulen et al. 1996 ; Schreuder et al. 1998 ). Although further observations are needed for a final conclusion, these data might suggest a possible different pathway of scrapie pathogenesis in heterozygous ARR sheep. Conversely, no PrPsc accumulation was detected in tissues from any of the sheep from the healthy control group (five ARR/ARR, two ARQ/ARR, and three ARQ/ARQ), in agreement with the histological examination of the obex. Therefore, contrary to recent reports (Ryder et al. 2001 ) correlated with labeling of residual PrPc in the obex, we observed no false-positive results. The MAb 2G11 used in this study does not, under our experimental conditions, label the ovine PrPc, generating no immunostaining in obex of sheep from scrapie-free flocks (Andreoletti et al. 2000 ).

Cell phenotyping using paraffin sections from ovine lymphoid tissues could not be carried out with classical anti-ovine CD antibodies because these require the use of frozen sections. Heterologous anti-CD antibodies were therefore selected and optimized for this purpose.

Ovine T- and mature B-lymphocytes were identified with antibodies (PAb A 0452 and MAb HM57, respectively) already known for their crossreactivity to several animal species and for their ability to work on paraffin sections (Jones et al. 1993 ). The present report shows that these antibodies also react with ovine antigens. Labeling of ovine T-cells with the anti-human CD3 PAb A 0452 on paraffin sections yielded a T-lymphocyte distribution comparable to that seen with the anti-ovine CD5 MAb SBU-T1 (Mackay et al. 1985 ) on frozen lymph node sections. Under our experimental conditions, the anti-human CD79{alpha}cy MAb HM57 appeared to label sheep immature B-cells only, the immunostaining of mature B-lymphocytes being achieved with the anti-human CD-20-like MAb BLA36. In humans these antibodies detect B-cell populations from the pre-B-cell stage throughout the maturation process, with disappearance of the CD20 expression at the plasmocyte stage. The difference that we observed between the labeling pattern of these two MAbs is probably due to the use of these antibodies in a heterologous system, combined with the effects of formalin fixation and paraffin embedding on tissue antigens. Nevertheless, anti-CD20-like and anti-CD79{alpha}cy antibodies allow a discriminative identification of the B-cell population.

Until now, no antibody specific for ovine FDC was available. Of the four antibodies tested, only MAb CNA.42 reacted with ovine FDC. This antibody, kindly provided by Dr. Delsol (CHU Purpan; Toulouse, France), binds to a formalin-resistant, 120-kD glycosylated antigen that has been characterized as a type IV myosin. In sheep and in most other mammalian species, this antigen appears to be selectively expressed in FDC and endothelial vascular cells (Raymond et al. 1997 ).

Identification of ovine macrophages in paraffin-embedded lymphoid tissue sections was carried out using the anti-human CD68 MAb Ki-M6. In humans, the CD68 antigen is expressed by monocytes, macrophages (Parwaresch et al. 1986 ), and microglial cells (Sasaki et al. 1993 ; Ulvestad et al. 1994 ). This antibody recognizes an intracytoplasmic lysosome-associated epitope resulting in a granular staining pattern. In sheep tissue sections, this antibody strongly labeled cells that, in lymphoid organs, are morphologically identical to macrophages and interdigitating dendritic cells and that are identical to microglial cells in the CNS. A more in-depth characterization of ovine CD68-positive cells is now being performed in our laboratory.

PrPsc deposition is considered to occur mainly in post-mitotic cells, such as neurons and FDCs (Fraser and Farquhar 1987 ; Bruce et al. 2000 ). We attempted to confirm this hypothesis by labeling proliferating cells in lymphoid tissues. In humans, dividing cells can be identified with antibodies specific for the nuclear Ki-67 antigen, which is expressed in all continuously cycling cells in G1-, S-, G2-, and M-phases but not in resting cells in G0 (Gerdes et al. 1984 ). The anti-Ki-67 MIB-1 MAb has already been characterized (Gerdes et al. 1983 ) and was used in different mammalian species, including sheep (Falini et al. 1989 ).

In the CNS, PAbs and MAbs raised against GFAP, S100 protein, synaptophysin, and neurofilament proteins are commonly used for identification of cell populations and structures in human brain (Miettinen et al. 1984 ; Wiedenmann and Franke 1985 ; McLendon and Bigner 1994 ). Most of these antibodies, specific for these phylogenetically conserved proteins, show broad interspecies crossreactivity and can therefore be used on ovine samples (Pegram et al. 1985 ; Duittoz et al. 1997 ). Among the commercially available reagents, we had previously selected the anti-GFAP PAb Z 0334, the anti-S100 protein PAb Z 0311, and the anti-synaptophysin MAb SY 38 to identify ovine astrocytes, neurons and synapses, respectively. Optimal immunostaining of the ovine neurofilament proteins was achieved with MAb 2F11, specific for the 70- and 200-kD subunits. This reagent constitutes a sensitive and accurate tool with which to study sheep neuronal processes.

This first step of antibody screening led to a selection of reagents suitable for identification of the main lymphoid and neural cell populations in ovine formalin-fixed and paraffin-embedded tissue samples. These antibodies, being largely unaffected by PrPsc IHC pretreatments, enabled us to set up a PrPsc double-labeling protocol.

Double-labeling methods described for PrPsc IHC comprise several steps and involve many antibody layers (Miyazono et al. 1991 ; Guiroy et al. 1994 ; Muhleisen et al. 1995 ; van Keulen et al. 1995 ). Each additional step may increase the risk for nonspecific labeling. The method we have developed and described here is based on the concurrent use of a pair of primary antibodies, each raised in a different animal species. This approach has allowed a significant reduction in the number of technical steps, making double-labeling as rapid as a single labeling technique.

In brain sections from clinically scrapie-affected animals, the PrPsc double-labeling technique has enabled us to localize PrPsc in microglial cells and astrocytes. These two cell subsets are already known to be involved in neuronal degeneration and cell death in prion diseases (Williams et al. 1997 ; Giese et al. 1998 ). Our method has also enabled us to more precisely locate PrPsc in neuronal structures. The presence of PrPsc in distal neuronal processes, as determined with the anti-neurofilament MAb 2F11, suggests PrPsc trafficking within neurons, as previously reported for PrPc (Rodolfo et al. 1999 ). This PrPsc trafficking may support the basis of the agent spread through neuroanatomic structures in the CNS. This hypothesis is also supported by the observation of PrPsc deposits in the vicinity of synaptophysin, indicating a possible synaptic location of the pathogenic protein and suggesting an inter-neuronal spread of the agent through these structures. PrPsc deposition in synaptic structures is in agreement with the proposed role of the PrPc protein in synaptic transmission (Collinge et al. 1994 ).

In lymphoid tissues, all our observations point to the role of non-proliferating macrophages (CD68-positive cells) and FDCs as target cells for the scrapie agent (Andreoletti et al. 2000 ; and this report), with no apparent involvement of lymphocyte subsets. Indeed, no PrPsc deposits could be detected in the cytoplasm of germinal center T- or B-lymphocytes, some granules being located in close contact with the outer surface of the plasma membrane of only few of them. However, these observations do not enable us to make a definite conclusion about a PrPsc extracellular deposition from its accumulation in the FDC network. In contrast to some studies in mice that suggested no direct involvement of bone marrow-derived cells in prion replication (Brown et al. 1999 ), our results emphasize a role for the macrophage lineage in the pathogenesis of sheep scrapie. Further investigations are required to better characterize this CD68-positive cell population in sheep lymphoid tissues (macrophages and/or interdigitating dendritic cells). These PrPsc-positive myeloid cells could represent a pathway for FDC infection and neuro-invasion (Andreoletti et al. 2000 ; Beekes and McBride 2000 ; Aucouturier et al. 2001 ) and/or may be involved in the clearance of the scrapie agent (Beringue et al. 2000 ).

In conclusion, this study led to the development of a straightforward and rapid PrPsc double-labeling technique allowing an accurate determination of the cell subsets and structures involved in the accumulation of PrPsc. The setting up of this new protocol involved screening of a number of antibodies that allowed cell phenotyping analysis in formalin-fixed and paraffin-embedded ovine tissues under the experimental conditions required for PrPsc IHC detection. This methodology constitutes a useful tool for further in situ investigations of the pathogenesis of scrapie and other ovine diseases.


  Acknowledgments

Supported by National and European grants (Interministerial Committee GIS "Prion diseases," Région Midi-Pyrénées, and the European programme FAIR CT 98-6013).

We thank Dr Lucien J.M. van Keulen (CIDC; Lelystad, The Netherlands), Dr Jeanne Grosclaude (Virologie et Immunologie Moléculaires, INRA; Jouy-en-Josas, France), Prof Georges Delsol (CHU Purpan; Toulouse, France), and Dr John Hopkins (Faculty of Veterinary Medicine, Summerhall; Edinburgh, Scotland) for supplying antibodies R521, 2G11, CNA.42, and VPM 65, respectively. We are grateful to Labogena (Jouy-en-Josas, France) for ovine PrP genotyping.

Received for publication September 5, 2001; accepted April 17, 2002.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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