1 Centre for Infectious Diseases, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK
2 Institute of Comparative Medicine, Department of Veterinary Pathology, University of Glasgow, Glasgow, UK
Correspondence
John Fazakerley
John.Fazakerley{at}ed.ac.uk
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ABSTRACT |
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Present address: Department of Pathology and Infectious Diseases, Royal Veterinary College, Hawkshead Lane, North Mymms, Hertfordshire AL9 7TA, UK.
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MAIN TEXT |
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CV mice were inoculated intracerebrally with either control (uninfected) brain homogenate or with brain homogenate from a ME7-infected, clinically affected C57BL mouse, as described previously (Scott & Fraser, 1984). Control and ME7-infected mice were perfused with RNase-free PBS prior to being killed at 40, 70, 100, 130, 160, 170, 180 and 210 days post-infection (p.i.) and at terminal disease (225235 days p.i.). Brains were removed and divided sagitally, with one-half of the brain being retained for RNA analysis and the other half being fixed [buffered formol saline or paraformaldehyde/lysineperiodate (PLP)], processed and embedded in paraffin wax. Further mice were perfused with 4 % paraformaldehyde for TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling) staining, PLP for PrP staining or PLP-containing glutaraldehyde for immunocytochemical studies of microglia.
Immunocytochemical studies were performed as described previously (Williams et al., 1994a, 1997a
), enabling the temporal progression of neuropathological features to be studied in detail (Fig. 1
). To ensure staining specificity, sections in which the specific primary antibody was replaced by the appropriate normal serum (for polyclonal antibodies) or PBS (for monoclonals) acted as controls. Some PrP staining, detected using the 4F2 (a gift from J. Grassi, CEA, France) and 6H4 (Prionics) antibodies, was seen as punctate intracellular immunoreactivity of occasional (rare), scattered neurons in control (uninfected) mice. In infected mice, additional PrP immunoreactivity in the hippocampus was detected first at 100 days p.i. and gradually increased thereafter. At 100 and 130 days p.i., this increased staining was detected as a diffuse extracellular staining, particularly in the molecular layer of the CA1 hippocampal region (Fig. 1A
); at later stages, more intense punctate extracellular deposits were evident (Fig. 1B
) and increased PrP immunoreactivity was distributed more widely throughout the hippocampus. Increased punctate staining was observed in the thalamus from 70 days p.i.; thereafter, the pattern of PrP staining in both the hippocampus and the thalamus was consistent with previous reports (Jeffrey et al., 2000
, 2001
). To assess glial cell activation, astrocytes were identified using a rabbit anti-cow GFAP (glial fibrillary acidic protein) antibody (Dako) and microglia using the monoclonal antibodies FA11 (anti-CD68), F4/80 (both obtained from Serotec) and TIB122 (anti-leukocyte common antigen), the hybridoma for which was obtained from ECC. Activation of both microglia and astrocytes, evaluated as intensity of staining and number of immunopositive cells, was detected first in the hippocampus at 100 days p.i. (Fig. 1C, E
), a time at which initial morphological changes in hippocampal neurons have been described previously (Jeffrey et al., 2000
). Initial microglial staining was apparent particularly when using the FA11 antibody. Resting microglia do not stain with this antibody. In the hippocampus, focal staining of microglia in the CA1 hippocampal region was observed first at 100 days p.i. (Fig. 1C
), although immunoreactivity was evident in the thalamus from 70 days p.i.. Thereafter, the extent of microglial activation, as identified by FA11 immunopositivity and by increased staining for the F4/80 and leukocyte common antigen, increased steadily in both intensity and distribution within the hippocampus until the terminal stages of disease (Fig. 1D
). Similarly, the intensity of GFAP staining within the hippocampus was slightly greater than control mice at 100 days p.i. (Fig. 1E
) and increased thereafter to show greatest intensity and numbers of GFAP-positive cells by the time of terminal disease (Fig. 1F
). These increases in glial cell immunostaining, indicating microglial and astrocyte activation, paralleled increases in PrP immunoreactivity. Initially (100 and 130 days p.i.), glial cell activation was seen as increased intensity of staining and increased number of immunopositive cells with a morphology similar to that of normal (resting) microglia and astrocytes, whereas at later time-points, increasing numbers of immunoreactive cells showed morphological changes with cell processes becoming reduced in number, broader and shorter and the cell body becoming more prominent and rounded. These morphological changes were consistent with a progressive increase in the extent of glial cell activation. None of the sections stained without primary antibody showed any specific immunoreactivity. Vacuolation within the hippocampus was seen first as scattered individual small vacuoles at 130 days p.i. and more frequent, larger vacuoles from 160 days p.i. onwards; again, this was consistent with previous reports (Jeffrey et al., 2000
). TUNEL staining, performed as described previously (Lucassen et al., 1995
; Williams et al., 1997a
), demonstrated that hippocampal CA1 pyramidal neuron loss was most evident at 170 days p.i. (Fig. 1G
), reflecting the known and approximately 50 % loss of these cells between 160 and 180 days p.i. in this model (Jeffrey et al., 2000
).
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To ensure that the low level of expression of specific cytokines observed in the scrapie-infected brain samples by RPA was not due to any degradation or inhibitor activity within these samples, the samples were used to measure the expression of transcripts expected to be present and elevated in the ME7-infected brain using a CNS inflammation probe set (ICAM-1, iNOS, A20, Mac-1, EB22/5.3, GFAP and L32), kindly provided by I. Campbell (Scripps Research Institute). At terminal disease, significant upregulation of GFAP (a marker of astrocytosis), Mac-1 (a marker for microglial activation) and EB22/5.3 (an acute-phase response gene shown previously to be elevated in the Chandler/SWRj scrapie model; Campbell et al., 1994) was detected readily (Table 1
), thus validating the RPA methodology.
Further analysis of IL-1, IL-1
and TNF
cytokine transcripts in ME7-infected brain was performed by quantitative real-time RT-PCR, the greater sensitivity of the assay enabling direct comparisons between transcript levels in control and infected samples. cDNA, prepared from RNA derived from control and terminal stage ME7-infected brain, was used as template in real-time PCR using the LightCycler Real-Time PCR system with the LightCycler FastStart DNA Master kit SYBR Green I (Roche Molecular Biochemicals), according to the manufacturer's instructions. Two pairs of nested PCR primers were designed for each of
-actin, IL-1
, IL-1
and TNF
. Serial dilutions of the first-round PCR product (generated using the external primer pair) acted as standards for second-round PCR, performed on the LightCycler using the internal primer pair. cDNA templates were normalized using
-actin and amplified alongside the appropriate standards and controls, enabling a relative quantification of transcript levels in control and infected samples. In accordance with RPA results, only in their IL-1
response did the terminal stage scrapie-infected mice show some degree of uniformity, with IL-1
being upregulated in all mice studied, with a 3·5-fold increase in mean transcript levels relative to controls (P<0·02, Student's t-test). At the group level, there was no significant difference between control and scrapie-infected mice in the levels of IL-1
and TNF
. However, at the level of individual mice, some infected mice did demonstrate reproducibly increased levels of IL-1
and TNF
, suggesting variability between individuals at terminal disease.
To determine whether the observed IL-1 gene upregulation was also apparent at the protein level, cryostat brain sections were stained with a sheep anti-mouse IL-1
antibody (NIBSC) and a peroxidase/anti-peroxidase method. IL-1
protein was not detectable at the earliest stages of microglial activation (100 and 130 days p.i.) but became detectable at low levels at 180 days p.i. as a few, weakly positive cells associated with the CA1 hippocampal region. Staining increased progressively thereafter until terminal disease when numerous, scattered, moderately positive immunoreactive glia were detectable in the hippocampus (Fig. 1H
), thalamus and, to a lesser extent, cerebral cortex. The morphology of many of the IL-1
-positive cells was consistent with that of astrocytes, again reflecting previous reports of IL-1 immunostaining in other murine scrapie models (Kordek et al., 1996
; Williams et al., 1994b
, 1997b
). These data are in good agreement with the first detection of transcripts at 180 days p.i. (Fig. 2A
) with a steady increase thereafter.
This study has demonstrated that induction of cytokines in the brains of ME7-infected mice is a late event, occurs at a low level and is restricted to IL-1, IL-1
, TNF
and TGF
1. Only IL-1
and TGF
1 were elevated consistently in scrapie-infected mice; however, their relationship, causative or consequent, to neuropathology remains unclear. The relatively early (180 days p.i.) upregulation of IL-1
makes it a more likely candidate for cause or contribution to disease than the late upregulation of TGF
1. Elevated IL-1
transcript is a consistent finding in other scrapie models (Campbell et al., 1994
; Kim et al., 1999
; Williams et al., 1994b
, 1997b
) and IL-1 receptor (type I)-deficient mice have an extended disease course following inoculation with the 139A scrapie agent (Baier et al., 2002
). Taken together, this evidence indicates that a role for IL-1
in TSE neuropathology cannot be ruled out. In contrast, current studies suggest that other proinflammatory cytokines, including IL-1
, IL-6 and TNF
, are unlikely to have any fundamental role in the initiation or development of neuropathology, since elevation of these transcripts was not detected or was only detectable at terminal disease and only in some mice. This is consistent with the finding that mice devoid of either IL-6 or TNF
do not show prolonged periods of incubation following intracerebral inoculation (Mabbott et al., 2000
).
A striking feature of the results obtained in this study was the limited extent of upregulation of the proinflammatory cytokines. Even IL-1, the predominant proinflammatory cytokine in this study, was only increased 3·5-fold in terminally affected animals relative to control animals. In contrast, the extent of gliosis in the ME7/CV scrapie model, described in detail here for the first time, was marked. The current study compared events in ME7-infected brains to those in SFV-infected SCID mouse brains. Despite glial cell activation being significantly less in virus-infected SCID mice than the marked activation observed in the scrapie-infected brain, upregulation of IL-1
, IL-1
, TNF
and LT
(as determined by RPA) was significantly greater. We conclude that the extent of cytokine induction in scrapie-infected brains is disproportionate to the level of gliosis, at least relative to events in a CNS virus infection.
A previous study of cytokine induction in ME7 infection of the related C57BL/6 mouse did not detect the upregulation of any proinflammatory cytokines (Walsh et al., 2001), whereas our study observed a slight increase in a limited number of cytokines. This difference may be due to the differing sensitivities of the techniques used in the two studies and the differing time-course of disease in these two mouse models. Furthermore, the study on C57BL/6 mice used stereotaxic intracerebral inoculation of small volumes of brain homogenate for infection, which may result in a different pathogenesis of disease to that generated by our conventional intracerebral inoculation. However, the current finding of TGF
1 upregulation at terminal disease is in agreement with that observed in the ME7/C57BL/6 model (Cunningham et al., 2002
).
In conclusion, our results suggest strongly that ILs 1, 2, 3, 4, 5, 6, 7, 10, 12 and 13, GM-CSF, IFN-
, LT
and TNF
have no role in neuropathological events in scrapie. IL-1
and TGF
1 were elevated consistently in scrapie-infected mice, with only IL-1
elevated prior to clinical disease, occurring around the time of hippocampal neuron loss. A direct role for IL-1
in TSE neuropathology cannot be excluded. However, it should be stressed that although IL-1
transcripts are elevated in scrapie-infected brain, their levels are disproportionately low relative to the extent of the glial cell response.
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ACKNOWLEDGEMENTS |
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Received 3 February 2003;
accepted 26 May 2003.