Michael A. Klein

A crucial role for B cells in neuroinvasive scrapie.

Although prions are most efficiently propagated via intracerebral inoculation, peripheral administration has caused kuru [Gajdusek et al, 1966], iatrogenic Creutzfeldt-Jakob disease (CJD) [Gibbs et al, 1997], bovine spongiform encephalitis (BSE), and new variant CJD [Hill et al, 1997; Bruce et al, 1997]. Neurological disease after peripheral inoculation depends on prion expansion within cells of the lymphoreticular system (LRS) [Lasmezas et al. 1996; Wilesmith et al, 1992]. In order to identify the nature of the latter cells, we inoculated a panel of immune deficient mice with prions intraperitoneally. While defects affecting only T lymphocytes had no apparent effect, all mutations affecting differentiation and responses of B lymphocytes prevented development of clinical scrapie. Since absence of B cells and of antibodies correlates with severe defects in follicular dendritic cells (FDCs), the lack of any of these three components may prevent clinical scrapie. Yet, mice expressing immunoglobulins exclusively of the M subclass without detectable specificity for PrPc, and mice with differentiated B cells but lacking functional FDCs, developed scrapie after peripheral inoculation: therefore, differentiated B cells appear to play a crucial role in neuroinvasion of scrapie regardless of B-cell receptor specificity.

A crucial role for B cells in neuroinvasive scrapie

Although prion proteins are most efficiently propagated through intracerebral inoculation, peripheral administration has caused the diseases kuru, iatrogenic Creutzfeldt–Jakob disease (CJD), bovine spongiform encephalopathy (BSE) and new-variant CJD,. The development of neurological disease after peripheral inoculation depends on prion expansion within cells of the lymphoreticular system,. Here we investigate the identity of these cells by using a panel of immune-deficient mice inoculated with prions intraperitoneally: we found that defects affecting only T lymphocytes had no apparent effect, but that all mutations that disrupted the differentiation and response of B lymphocytes prevented the development of clinical scrapie. As an absence of B cells and of antibodies correlates with severe defects in follicular dendritic cells, a lack of any of these three components may prevent the development of clinical scrapie. However, we found that scrapie developed after peripheral inoculation in mice expressing immunoglobulins that were exclusively of the M subclass and without detectable specificity for the normal form of the prion PrPc, and in mice which had differentiated B cells but no functional follicular dendritic cells. We conclude that differentiated B cells are crucial for neuroinvasion by scrapie, regardless of the specificity of their receptors.

Complement facilitates early prion pathogenesis

New-variant Creutzfeldt–Jakob disease and scrapie are typically initiated by extracerebral exposure to the causative agent, and exhibit early prion replication in lymphoid organs. In mouse scrapie, depletion of B-lymphocytes prevents neuropathogenesis after intraperitoneal inoculation, probably due to impaired lymphotoxin-dependent maturation of follicular dendritic cells (FDCs), which are a major extracerebral prion reservoir. FDCs trap immune complexes with Fc-γ receptors and C3d/C4b-opsonized antigens with CD21/CD35 complement receptors. We examined whether these mechanisms participate in peripheral prion pathogenesis. Depletion of circulating immunoglobulins or of individual Fc-γ receptors had no effect on scrapie pathogenesis if B-cell maturation was unaffected. However, mice deficient in C3, C1q, Bf/C2, combinations thereof or complement receptors were partially or fully protected against spongiform encephalopathy upon intraperitoneal exposure to limiting amounts of prions. Splenic accumulation of prion infectivity and PrPSc was delayed, indicating that activation of specific complement components is involved in the initial trapping of prions in lymphoreticular organs early after infection.

PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain

Much available evidence points to a pathological isoform of the prion protein PrP being the infectious agent that causes transmissible spongiform encephalopathies, but the mechanisms controlling the neurotropism of prions are still unclear. We have previously shown that mice that do not express PrP (Prnp[o/o] mice) are resistant to infection by prions, and that if a Prnp(+/+) neurograft is introduced into such animals and these are infected intracerebrally with scrapie, the graft but not the surrounding tissue shows scrapie pathology. Here we show that PrP-expressing neurografts in Prnp(o/o) mice do not develop scrapie histopathology after intraperitoneal or intravenous inoculation with scrapie prions. Prion titres were undetectable in spleens of inoculated Prnp(o/o) mice, but were restored to wild-type levels upon reconstitution of the host lymphohaemopoietic system with PrP-expressing cells. Surprisingly, however, i.p. or i.v. inoculation failed to produce scrapie pathology in the neurografts of 27 out of 28 reconstituted animals, in contrast to intracerebral inoculation. We conclude that transfer of infectivity from the spleen to the central nervous system is crucially dependent on the expression of PrP in a tissue compartment that cannot be reconstituted by bone marrow transfer. Thus the requirement for the normal isoform of PrP in peripheral tissues represents a bottleneck for the spread of prions from peripheral sites to the central nervous system.

PrP expression in B lymphocytes is not required for prion neuroinvasion

Prion diseases are typically initiated by infection of peripheral sites, as in the case of bovine spongiform encephalopathy, new variant Creutzfeldt-Jakob disease, kuru and most cases of iatrogenic Creutzfeldt-Jakob disease. In mouse scrapie, prion infectivity accumulates in lymphoid organs, and the absence of mature B lymphocytes prevents peripherally administered prions from inducing central nervous system disease. We have now assessed whether expression of the cellular prion protein, PrPC, is required for B lymphocytes to mediate neuroinvasion. We found that repopulation of SCID and Rag-1-/- mice with fetal liver cells from either PrP-expressing or PrP-deficient mice and from T-cell deficient mice, but not from B-cell deficient mice, is equally efficient in restoring neuroinvasion after intraperitoneal inoculation of scrapie prions. These results indicate that cells whose maturation depends on B cells or their products, such as follicular dendritic cells, may enhance neuroinvasion. Alternatively, B cells may transport prions to the nervous system by a PrP-independent mechanism.

Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain

PrP knockout mice in which only the open reading frame was disrupted (‘Zürich I’) remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, Prnd. A new PrP knockout line, ‘Zürich II’, with a 2.9 kb Prnp deletion, developed this phenotype at ∼10 months (50% morbidity). A single Prnp allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a Prnd‐containing cosmid expressed Dpl at twice the level and became ataxic ∼5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.

Transepithelial prion transport by M cells

To the editor—The appearance of a new variant of Creutzfeldt–Jakob disease has raised concerns that bovine spongiform encephalopathies might be communicable to humans by dietary exposure. Membranous epithelial cells (M cells) are key sites of antigen sampling for the mucosa-associated lymphoid system and have been recognized as major ports of entry for enteric pathogens via transepithelial transport. Recently, in vitro systems have been developed in which epithelial cells undergo differentiation to cells resembling M cells by morphological and functional-physiological criteria. Here we investigated whether M cells are a plausible site of prion entry in a coculture model. We seeded 3 × 10 Caco-2 cells (clone TC7) on the upper face of 6.5-mm filters (3μm pore Transwell filters, Costar, Cambridge, Massachusetts) and cultured in culture medium (high-glucose DMEM supplemented with 10% FCS) for 72 h until confluency was reached. Next, we placed 1 × 10 Raji B cells diluted in 80 μl low-glucose culture medium onto the lower chamber facing the basolateral side of the Caco-2 cells by inverting the inserts. Lymphoid cells migrated through the pores of the filter and settled within the epithelial monolayer (Fig. 1a–c), inducing differentiation of some Caco-2 cells into M cells. Raji B cells were exclusively found at the basolateral site within Caco-2 monolayers (Fig. 1c), most likely because Raji cells express the chemokine receptor CCR6 that responds to CCL20, a chemokine secreted basolaterally by Caco-2 cells. We ascertained successful conversion by monitoring active, temperature-dependent transport of inert FITC-conjugated latex beads (FluoSpheres, 0.2 μm, Molecular Probes, Eugene, Oregon). Introduction of beads in the chamber facing the apical surface of epithelial cells allowed fluorescence recovery from the opposite compartment in confluent (as routinely analyzed by transepithelial resistance (TER) of ∼ 200 Ω × cm or occasionally by [H]inulin permeability) and M-cell–containing cocultures at 37 °C, as examined by FACS analysis (Fig. 1d). In contrast, there was no transepithelial transport of beads at 4 °C (data not shown) or in Caco-2 monolayers at 37 °C. Cocultures that combined integrity and active transport of beads (∼ 10% of cocultures) were incubated with 5 or 3 logLD50 Rocky Mountain Laboratory scrapie strain prions administered to the apical compartment. After 24 h, infectivity within the basolateral compartment was determined by bioassay with tga20 mice, which overexpress a Prnp transgene and develop scrapie rapidly after infection. Upon challenge with 5 logLD50 scrapie prions, we consistently recovered prions in the basolateral compartment of cocultures containing M cells (n = 3), indicating transepithelial prion transport (Fig. 1e). Even at very low prion doses (3 logLD50), we found infectivity in at least one M-cell–containing coculture (n = 3) Transepithelial prion transport by M cells

Fas Ligand Expression in Glioblastoma Cell Lines and Primary Astrocytic Brain Tumors

Fas/APO‐1 (CD95) is a cell surface receptor that mediates apoptosis when it reacts with Fas ligand (FasL) or Fas antibody. We previously reported that Fas expression is predominantly induced in perinecrotic glioma cells, suggesting that Fas induction is associated with apoptosis and necrosis formation, a histological hallmark of glioblastomas. In this study, we assessed the expression of FasL in 10 glioblastoma cell lines and in 14 astrocytic brain tumors (three low‐grade astrocytomas and 11 glioblastomas). Reverse transcriptase (RT)‐PCR revealed that all glioblastoma cell lines and primary astrocytic brain tumors express FasL. Immunohistochemically, FasL was predominantly expressed on the plasma membrane of glioma cells. These results suggest that FasL expression is common in human astrocytic brain tumors and may cause apoptosis of glioma cells if Fas expression is induced.

Lymph nodal prion replication and neuroinvasion in mice devoid of follicular dendritic cells

Variant Creutzfeldt–Jakob disease and scrapie are typically initiated by extracerebral exposure to prions, and exhibit early prion accumulation in germinal centers. Follicular dendritic cells (FDCs), whose development and maintenance in germinal centers depends on tumor necrosis factor (TNF) and lymphotoxin (LT) signaling, are thought to be indispensable for extraneural prion pathogenesis. Here, we administered prions intraperitoneally to mice deficient for TNF and LT signaling components. LTα−/−, LTβ−/−, LTβR−/−, and LTα−/− × TNFα−/− mice resisted infection and contained no infectivity in spleens and lymph nodes (when present). However, TNFR1−/−, TNFR2−/−, and some TNFα−/− mice developed scrapie similarly to wild-type mice. High prion titers were detected in lymph nodes, but not spleens, of TNFR1−/− and TNFα−/− mice despite absence of FDCs and germinal centers. Transfer of TNFR1−/− fetal liver cells into lethally irradiated Prnp0/0 mice restored infectivity mainly in lymph nodes. Prion protein (PrP) colocalized with a minority of macrophages in tumor necrosis factor receptor (TNFR) 1−/− lymph nodes. Therefore, prion pathogenesis can be restricted to lymphoreticular subcompartments, and mature follicular dendritic cells are dispensable for this process. Macrophage subsets are plausible candidates for lymphoreticular prion pathogenesis and neuroinvasion in the absence of FDCs, and may represent a novel target for postexposure prophylaxis.

Lymphatic Dissemination and Comparative Pathology of Recombinant Measles Viruses in Genetically Modified Mice

ABSTRACT The dissemination of the Edmonston measles virus (Ed-MV) vaccine strain was studied with genetically modified mice defective for the alpha/beta interferon receptor and expressing human CD46 with human-like tissue specificity and efficiency. A few days after intranasal infection, macrophages expressing Ed-MV RNA were detected in the lungs, in draining lymph nodes, and in the thymus. In lymph nodes, large syncytia which stained positive for viral RNA and for macrophage surface marker proteins were found and apoptotic cell death was monitored. In the thymus, smaller syncytia which stained positive for macrophage and dendritic cell markers were detected. Thus, macrophages appear to be the main vectors for dissemination of MV infection in these mice; human macrophages may have a similar function in the natural host. We then compared the pathogenicities of two recombinant viruses lacking the C or V nonstructural proteins to that of the parental strain, Ed-MV. These viruses were less effective in spreading through the lymphatic system and, unlike Ed-MV, were not detected in the liver. After intracerebral inoculation the recombinant viruses caused lethal disease less often than Ed-MV and induced distinctive patterns of gliosis and inflammation. Ed-MV was reisolated from brain tissue, but its derivatives were not. C- and V-defective viruses should be considered as more-attenuated MV vaccine candidates.