Karen L. Brown

Sites of prion protein accumulation in scrapie-infected mouse spleen revealed by immuno-electron microscopy

Prion protein (PrP) from the brains of animals with transmissible spongiform encephalopathies is partially protease resistant (PrPres) compared with fully sensitive PrP (PrPsen) from uninfected brains. In most experimental models, PrPres is a reliable indicator of infectivity. Light microscopic studies have suggested that both PrPsen and disease‐specific accumulations of PrP are associated with follicular dendritic cells (FDCs). Using immunogold electron microscopy, this study has demonstrated disease‐specific accumulation of PrP in the spleens of C57 BL mice, 70 days after intracerebral infection with the ME7 strain of scrapie and at the terminal stage of disease at 170 days. At both stages, tingible body macrophages contained PrP within lysosomes and PrP was also detected at the plasmalemma of FDCs. In the light zone of follicles of terminally diseased mice, all FDC dendrites were arranged in the form of highly reactive or hyperplastic labyrinthine glomerular complexes, within which PrP was consistently seen between FDC processes in association with abundant electron dense material, interpreted as antigen–antibody complexes. Within some glomeruli, fibrillar forms of PrP consistent with amyloid were seen. At 70 days after challenge, large or hyperplastic labyrinthine complexes were rare and invariably labelled for PrP. However, sparse PrP labelling was also seen on simple FDC processes at this stage. The ubiquitous accumulation of extracellular PrP in complex glomerular dendrites of FDCs in spleens from terminally affected mice, contrasted with simple FDC profiles, sparse PrP and limited electron dense deposits in all but a few FDCs of 70‐day post‐infected mice. This suggests that FDCs continually release PrP from the cell surface, where it is associated with trapped antigen–antibody complexes and dendritic extension. It is likely that tingible body macrophages acquire PrP following phagocytosis of PrP within iccosomes or from the extracellular space around FDC dendrites. These studies would not support an intracellular phase of PrP accumulation in FDCs but show that PrP is produced in excess by scrapie‐infected cells from where it is released into the extracellular space. We suggest that PrPsen is involved in dendritic extension or in the process of antibody–antigen trapping, perhaps as part of the binding mechanism for antigen–antibody complexes.

T‐lymphocyte activation and the cellular form of the prion protein

The transmissible spongiform encephalopathies are neurodegenerative disorders which include Creutzfeldt–Jakob disease in humans, and scrapie and bovine spongiform encephalopathy in animals. A major component of the infectious agent responsible for these diseases is considered to be a post‐translationally modified form of a host‐encoded glycoprotein PrPc, termed PrPSc. While PrPc is abundantly expressed in tissues of the central nervous system (CNS), little is known about its normal function. The expression of PrPc is not restricted to the CNS, as this protein can also be detected in the lymphoid tissues of mice and sheep. In this report we demonstrate that resting murine splenic lymphocytes express PrPc protein on their cell membranes. Furthermore, expression of PrPc was significantly enhanced following in vitro stimulation with the non‐specific T‐cell mitogen concanavalin A (Con A). Genetically engineered mice with an inactive PrPc gene (PrP−/− mice), were utilized to investigate the involvement of PrPc in lymphocyte activation. Experiments revealed that the Con A‐induced proliferation of lymphocytes from PrP−/− mice was significantly reduced to ≈50–80% that of wild‐type (PrP+/+) mice 48 hr post‐stimulation. These findings demonstrate an important role for PrPc in extra‐neuronal tissues and suggest that PrPc is a lymphocyte surface molecule that participates in T‐cell activation.

Replication of scrapie in spleens of SCID mice follows reconstitution with wild-type mouse bone marrow

SCID mice are resistant to intraperitoneal infection with 10(3) and 10(4) intracerebral ID50 units of ME7 scrapie agent whereas they develop disease after intracerebral challenge. However, higher doses introduced, by intraperitoneal or subcutaneous routes, produce disease. Immunocompetent mice of the same strain (CB20) developed scrapie following either intracerebral or intraperitoneal infection. Bioassay of spleens from SCID mice infected with 10(-1) dilutions of ME7 scrapie by intraperitoneal, intracerebral or abdominal subcutaneous injection showed traces or low levels of infectivity in spleen. However, subcutaneous injection beneath the skin of the neck failed to infect the spleen. CB20 bone marrow reconstitution of SCID mice resulted in the regeneration of a normal lymphoid architecture in the spleen. Spleens from these reconstituted mice, infected intracerebrally with a 10(-1) dilution of ME7 contained high levels of infectivity. These results suggest that the ability to replicate scrapie agent in spleen or lymphoid tissue depends on the restoration of normal lymphoid structure and in particular the presence of differentiated follicular dendritic cells. The possibility that SCID mice can select minor strains of scrapie which are normally unrecognized in cloned ME7 is discussed.

Follicular dendritic cells in TSE pathogenesis

The pathogenesis of transmissible spongiform encephalopathies (TSEs) often includes a replication phase in lymphoid tissues before infection spreads to the central nervous system. Recent studies show that the follicular dendritic cells of the germinal centres are critical for this replication. These cells are therefore potential targets for therapy or prophylaxis in natural TSEs, such as variant Creutzfeldt-Jakob disease.

Follicular Dendritic Cell-Specific Prion Protein (PrPc) Expression Alone Is Sufficient to Sustain Prion Infection in the Spleen

Prion diseases are characterised by the accumulation of PrPSc, an abnormally folded isoform of the cellular prion protein (PrPC), in affected tissues. Following peripheral exposure high levels of prion-specific PrPSc accumulate first upon follicular dendritic cells (FDC) in lymphoid tissues before spreading to the CNS. Expression of PrPC is mandatory for cells to sustain prion infection and FDC appear to express high levels. However, whether FDC actively replicate prions or simply acquire them from other infected cells is uncertain. In the attempts to-date to establish the role of FDC in prion pathogenesis it was not possible to dissociate the Prnp expression of FDC from that of the nervous system and all other non-haematopoietic lineages. This is important as FDC may simply acquire prions after synthesis by other infected cells. To establish the role of FDC in prion pathogenesis transgenic mice were created in which PrPC expression was specifically “switched on” or “off” only on FDC. We show that PrPC-expression only on FDC is sufficient to sustain prion replication in the spleen. Furthermore, prion replication is blocked in the spleen when PrPC-expression is specifically ablated only on FDC. These data definitively demonstrate that FDC are the essential sites of prion replication in lymphoid tissues. The demonstration that Prnp-ablation only on FDC blocked splenic prion accumulation without apparent consequences for FDC status represents a novel opportunity to prevent neuroinvasion by modulation of PrPC expression on FDC.

Involvement of the immune system in TSE pathogenesis

Abstract The transmissible spongiform encephalopathies (TSEs) are continuing to attract both scientific interest and public concern. Here, Neil Mabbott and colleagues review current understanding of the peripheral pathogenesis of TSE diseases.

The Effects of Host Age on Follicular Dendritic Cell Status Dramatically Impair Scrapie Agent Neuroinvasion in Aged Mice

Following peripheral exposure, many transmissible spongiform encephalopathy (TSE) agents accumulate first in lymphoid tissues before spreading to the CNS (termed neuroinvasion) where they cause neurodegeneration. Early TSE agent accumulation upon follicular dendritic cells (FDCs) in lymphoid follicles appears critical for efficient neuroinvasion. Most clinical cases of variant Creutzfeldt-Jakob disease have occurred in young adults, although the reasons behind this apparent age-related susceptibility are uncertain. Host age has a significant influence on immune function. As FDC status and immune complex trapping is reduced in aged mice (600 days old), we hypothesized that this aging-related decline in FDC function might impair TSE pathogenesis. We show that coincident with the effects of host age on FDC status, the early TSE agent accumulation in the spleens of aged mice was significantly impaired. Furthermore, following peripheral exposure, none of the aged mice developed clinical TSE disease during their lifespans, although most mice displayed histopathological signs of TSE disease in their brains. Our data imply that the reduced status of FDCs in aged mice significantly impairs the early TSE agent accumulation in lymphoid tissues and subsequent neuroinvasion. Furthermore, the inefficient neuroinvasion in aged individuals may lead to significant levels of subclinical TSE disease in the population.

Alterations in junctional proteins, inflammatory mediators and extracellular matrix molecules in eosinophilic esophagitis

Eosinophilic esophagitis (EoE), an inflammatory atopic disease of the esophagus, causes massive eosinophil infiltration, basal cell hyperplasia, and sub-epithelial fibrosis. To elucidate cellular and molecular factors involved in esophageal tissue damage and remodeling, we examined pinch biopsies from EoE and normal pediatric patients. An inflammation gene array confirmed that eotaxin-3, its receptor CCR3 and interleukins IL-13 and IL-5 were upregulated. An extracellular matrix (ECM) gene array revealed upregulation of CD44 & CD54, and of ECM proteases (ADAMTS1 & MMP14). A cytokine antibody array showed a marked decrease in IL-1α and IL-1 receptor antagonist and an increase in eotaxin-2 and epidermal growth factor. Western analysis indicated reduced expression of intercellular junction proteins, E-cadherin and claudin-1 and increased expression of occludin and vimentin. We have identified a number of novel genes and proteins whose expression is altered in EoE. These findings provide new insights into the molecular mechanisms of the disease.

Severely combined immunodeficient (SCID) mice resist infection with bovine spongiform encephalopathy

Following combined intraperitoneal and intracerebral injection with bovine spongiform encephalopathy (BSE) cow brain homogenate, SCID mice show a resistance to infection in comparison with immunocompetent CB20 mice. BSE occurred in only five out of 22 challenged SCID mice, with a mean incubation period of 573 days, whereas all the CB20 mice developed the disease with a mean incubation period of 456 days. In contrast, previous studies have shown that intracerebral infection of SCID mice with a mouse-passaged scrapie strain, ME7, produces 100% incidence of disease but no replication of infectivity in spleen. The results with BSE suggest that there is little or no direct infection of the CNS in interspecies transmissions, but that processing or replication of infectivity in peripheral lymphoid tissues may facilitate subsequent spread of infection to the CNS.

Glycosylation of PrPC Determines Timing of Neuroinvasion and Targeting in the Brain following Transmissible Spongiform Encephalopathy Infection by a Peripheral Route

ABSTRACT Transmissible spongiform encephalopathy (TSE) infectivity naturally spreads from site of entry in the periphery to the central nervous system where pathological lesions are formed. Several routes and cells within the host have been identified as important for facilitating the infectious process. Expression of the glycoprotein cellular PrP (PrPC) is considered a key factor for replication of infectivity in the central nervous system (CNS) and its transport to the brain, and it has been suggested that the infectious agent propagates from cell to cell via a domino-like effect. However, precisely how this is achieved and what involvement the different glycoforms of PrP have in these processes remain to be determined. To address this issue, we have used our unique models of gene-targeted transgenic mice expressing different glycosylated forms of PrP. Two TSE strains were inoculated intraperitoneally into these mice to assess the contribution of diglycosylated, monoglycosylated, and unglycosylated PrP in spreading of infectivity to the brain. This study demonstrates that glycosylation of host PrP has a profound effect in determining the outcome of disease. Lack of diglycosylated PrP slowed or prevented disease onset after peripheral challenge, suggesting an important role for fully glycosylated PrP in either the replication of the infectious agent in the periphery or its transport to the CNS. Moreover, mice expressing unglycosylated PrP did not develop clinical disease, and mice expressing monoglycosylated PrP showed strikingly different neuropathologic features compared to those expressing diglycosylated PrP. This demonstrates that targeting in the brain following peripheral inoculation is profoundly influenced by the glycosylation status of host PrP.