ARTICLE |
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
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Summary |
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Transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by amyloid deposition of proteinprion (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 50:13571370, 2002)
Key Words: PrPsc double labeling, immunohistochemistry, ovine scrapie
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Introduction |
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The pathogenesis of transmissible spongiform encephalopathies (TSEs), such as scrapie in sheep (
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 (
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 (
Neurodegeneration in TSEs is characterized by vacuolar changes of the neuropil and neurons in the gray matter, gliosis, and neuronal loss (
The disease-specific PrPsc accumulations are mainly composed of deposits in the B-follicle germinal centers of lymphoid formations (
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 (
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 (
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.
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Materials and Methods |
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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 410 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;
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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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 (
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Antigen Retrieval Protocols
Our antigen retrieval protocol comprised a mild proteolysis followed by hydrated buffered autoclaving (
PrPsc-specific labeling requires an initial formic acid treatment before the antigen retrieval steps (
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 streptavidinperoxidase 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 phosphatasestreptavidin 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 510 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 320 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.
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Results |
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PrPsc Immunolabeling
The MAb 2G11 and PAb R521 gave similar results, as previously described (
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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Among the antibodies tested to identify ovine B-lymphocytes, only two, MAb BLA36 (anti-CD20-like) (Fig 1C) and MAb HM57 (anti-CD79cy) (Fig 1E), allowed specific labeling of these cells. Using the anti-CD79
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 (
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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 (
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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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 (CD79cy-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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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).
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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 (
Recent reports have described new methodologies applied to prionprotein IHC. Ovine PrPsc was specifically detected in frozen lymphoid tissue sections (
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 (
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 (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
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 (
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 (
PrPsc deposition is considered to occur mainly in post-mitotic cells, such as neurons and FDCs (
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 (
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 (
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 (
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 (
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.
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Acknowledgments |
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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.
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