News Physiol Sci 15: 250-255, 2000;
1548-9213/00 $5.00
News in Physiological Sciences, Vol. 15, No. 5, 250-255,
October 2000
© 2000 Int. Union Physiol. Sci./Am. Physiol. Soc.
The Neuroimmune Interface in Prion Diseases
Michael A. Klein and
Adriano Aguzzi
M. A. Klein and A. Aguzzi are at the Institute of Neuropathology, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland.
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Abstract
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Prion diseases are fatal neurodegenerative disorders of animals and humans. Here we address the role of the immune system in the spread of prions from peripheral sites to the central nervous system and its potential relevance to iatrogenic prion disease.
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Introduction
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Prion diseases or transmissible spongiform encephalopathies belong to a group of fatal neurodegenerative illnesses that includes Creutzfeldt-Jakob Disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle (1, 11). Experimental and epidemiological investigations of prion diseases have been greatly spurred by the recognition of new variant CJD (vCJD) in the United Kingdom and in France and by the subsequent demonstration that bovine prions can be experimentally transmitted to a variety of species, including humans, by the consumption of beef products contaminated with BSE (4).
Prion diseases are characterized by the deposition of PrPSc, an abnormal, relatively protease-resistant isomer of a normal host-encoded cellular glycoprotein called PrPC. Prion infectivity copurifies with PrPSc, which suggests that this abnormal isomer is a component of the infectious agent (11). Mice deficient in PrPC (Prnpo/o) fail to develop scrapie and do not propagate the infectious agent, demonstrating that host cells must express PrPC to sustain the disease (3). Although PrPC is expressed at high levels in the central nervous system (CNS), it is not confined to this site and can be detected on a variety of cells in peripheral tissues, including cells of the lymphoid system.
Prnpo/o mice develop quite normally, at least for the first 70 wk of their typical life span of 100 wk (13). Such mice do show subtle aberrations, such as impaired 
-aminobutyric acid type A receptor-mediated fast inhibition and long-term potentiation in the hippocampus and altered circadian rhythms, but they appear to be clinically normal and are able to reproduce. As they age, however, prion-deficient mice of one strain generated in Japan (but not of the strains generated in Zurich or in Edinburgh) show progressive signs of ataxia that lead to premature death (13), although this might be a consequence of additional deletions in these strains (15). Furthermore, speculations were raised that the prion protein may somehow be involved in certain senile dementias (11), with or without additional cofactors.
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Peripheral route of infection
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Although the pathology of prion diseases is confined mainly to the brain and can occur following iatrogenic intracerebral inoculation, the most common occurrence of disease results from exposure via peripheral routes, such as intraperitoneal, intravenous, or oral exposure to infectious material. Following either intracerebral or intraperitoneal experimental inoculation, prions accumulate in secondary lymphoid organs such as spleen and lymph nodes (6). This appears to be a strain-dependent phenomenon, since different prion strains exhibit different affinities for lymphoid tissues. For example, BSE appears to have low affinity for lymphoid tissue of cow and is confined to the nervous system of experimentally infected cattle, although very limited amounts of infectivity have been detected in other sites, such as terminal ileum and bone marrow. In contrast, during the human disease vCJD, which is most probably derived from BSE, PrPSc accumulates in tonsils, spleen, and appendix of infected individuals.
Following experimental peripheral prion inoculation of mice, there is a typically prolonged, clinically silent phase during which prions replicate within the lymphoreticular system. This occurs before detectable neuroinvasion by prions and the subsequent occurrence of neurological symptoms. During this preclinical period, prions may replicate to high titers within lymphoreticular tissues. Elucidating which cells within the peripheral lymphoid tissue support prion replication and, crucially, how prions are transported to the CNS is a major scientific goal and of considerable clinical importance. The widespread exposure of the UK population and those of other countries to BSE has led to concerns of a potential human prion disease epidemic. It is now clear that the 42 confirmed cases of vCJD in the UK, France, and Ireland are caused by a prion strain that shares considerable identity to that which has caused BSE in cattle.
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The interplay between B cells and follicular dendritic cells in prion neuroinvasion
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Despite considerable evidence implicating the role of the immune system in peripheral prion pathogenesis, there have been few studies on the identity of cells involved in this process. Early studies showed that whole body gamma irradiation of mice failed to influence prion pathogenesis or scrapie incubation time (5). This has argued against a significant involvement by proliferating cells in the lymphoreticular phase of prion propagation. Follicular dendritic cells (FDC), which are radio resistant, have been considered the prime cell type for prion replication within lymphoid tissue because PrPSc accumulates in the follicular dendritic network of scrapie-infected wild-type and nude mice (7). In addition, severe combined immunodeficient mice (SCID), which lack mature B and T cells, and which do not appear to have functional FDCs, are highly resistant to scrapie after intraperitoneal inoculation and fail to replicate prions in the spleen. Interestingly, bone marrow reconstitution of SCID mice with wild-type spleen cells restores their susceptibility to scrapie disease after peripheral infection (10). These findings suggest that an intact or partially intact immune system, comprising lymphocytes and FDCs, is required for efficient neuroinvasion by prions from the site of peripheral infection.
The time course and the susceptibility to the development of scrapie disease following intracerebral or intraperitoneal inoculation is highly reproducible and is dependent primarily on the dose of the inoculum. Therefore, neuroinvasion by prions migrating from peripheral lymphoid tissue may depend on controlled, rate-limiting reactions. To identify such rate-limiting steps during prion neuroinvasion, PrPC-deficient mice bearing PrP-overexpressing cerebral neurografts were constructed and infected intraperitoneally. No disease was observed within the cerebral grafts, suggesting that neuroinvasion depends on PrP expression in extracerebral sites, including neurons. This was further underlined by reconstitution of the lymphoid system with PrPC-expressing cells, which restored infectivity in the lymphoid tissue but still failed to transport prions to the nervous system (2).
To identify the lymphoid cells responsible for accumulation of the infectious agent in secondary lymphoid organs, we have investigated experimental prion disease in a panel of immunodeficient mice following peripheral and intracerebral inoculation with Rocky Mountains Laboratory inocula. The panel of immunodeficient mice used comprised some that lacked both B and T cells (RAG-1–/–, RAG-2–/–, and SCID mice) as well as AGR–/– mice that lacked the receptors for interferon-
/-ß and interferon- in addition to B and T cells. The role of T cells in peripheral prion disease was investigated with the use of mice that lacked subsets of these cells (CD4–/–, CD8–/–, and ß2-microglobulin–/– mice) or lacked expression of the cytotoxic molecule perforin (PKOB mice). The role of B cells was studied in µMT–/– mice, which have a targeted disruption of the transmembrane exon of the immunoglobulin µ-chain gene. These mice do not produce any immunoglobulins and suffer from a B cell differentiation block at the large-to-small pre-B cell transition yet bear complete and functional T cell subsets.
Intracerebral challenge of each strain of immune-deficient mice with scrapie prions resulted in the development of clinical symptoms of disease with a comparable time course to that seen in wild-type mice (Fig. 1A
). Disease was confirmed by histopathological analysis, Western blot, and transmission of disease to tga20 indicator mice, which overexpress the normal prion protein (PrPC) and are hypersensitive to mouse prions. We concluded from this part of the study that after prions have gained access to the nervous system, prion expansion in the brain and scrapie pathogenesis proceed without any detectable influence due to the immune status of the host.
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FIGURE 1. Latency of scrapie in different immunodeficient mice. All mice developed spongiform encephalopathy after intracerebral inoculation (A; closed triangles). In contrast, B cell deficient mice stayed healthy after intraperitoneal inoculation of RML scrapie prions (B; open circles).
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In contrast, after intraperitoneal inoculation of the panel of immunodeficient mice, no clinical disease was observed in mice with either a B cell defect or with a combined B and T cell deficiency (Fig. 1B
). Importantly, no prion infectivity was detectable in the spleens of disease-free mice. In SCID mice, which also lack B and T lymphocytes, scrapie disease was marginally prolonged after intraperitoneal challenge. This may be due to an incomplete immune deficiency in SCID mice on a C57BL/6 genetic background. Mice with a T cell defect exposed to prions via the intraperitoneal route developed scrapie disease.
These data implicate B cells as a critical cell type involved in peripheral scrapie pathogenesis. However, in the absence of B cells mice fail to produce antibodies and FDCs fail to develop. To distinguish which of these three factors may be responsible for neuroinvasion by prions, two further mouse strains were investigated. To elucidate the role of immunoglobulins, we analyzed mice producing antibody exclusively of the IgM subclass (t11µMT) and that had no detectable specificity for PrPC. The role of FDCs was addressed using mice that lacked functional FDCs (TNFR1–/–) but have differentiated B cells. Both strains of mice developed scrapie after peripheral inoculation with RML inocula, demonstrating a crucial role for differentiated B cells per se in neuroinvasion of scrapie (8).
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Expression of PrPC on B lymphocytes is not required for prion neuroinvasion
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Since the replication of prions (3) and their transport from the periphery to the CNS (2) is dependent on expression of PrPC, we examined whether expression of PrPC by B cells was necessary to support neuroinvasion. Mice with various immune defects were repopulated by adoptive transfer of hematopoietic stem cells, which expressed or lacked expression of PrPC.
Adoptive transfer of either Prnp+/+ or Prnpo/o fetal liver cells (FLCs) induced formation of germinal centers in spleens of recipient mice and differentiation of FDCs, as visualized by staining with antibody FDC-M1 (Fig. 3). However, no FDCs were found in B and T cell-deficient mice reconstituted with FLCs from µMT embryos (B cell deficient), consistent with the notion that B cells or products thereof are required for FDC maturation.
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FIGURE 3. Histology of spleens from Rag-1–/– mice reconstituted with Prnpo/o FLCs. Top: in paraffin sections stained with hemalaun before FLC transfer (left), no B cell follicles or germinal centers were discernible in Rag-1–/– mice; restoration of organized B cell follicles and germinal centers after FLC reconstitution are shown at middle (magnification, x200). Frozen section immunostained with follicular dendritic cell (FDC) antibody FDC-M1 revealed formation of prominent FDC clusters within germinal centers after FLC transfer (right; magnification x250). Bottom: confocal double-color immunofluorescence analysis of splenic germinal centers in Rag-1–/– mice reconstituted with Prnpo/o FLCs after intraperitoneal inoculation with RML prions. Sections were stained with antibody FDC-M1 to FDCs (green; left) and with antiserum R340 to PrP (red; middle). Regions in which both signals are detectable appear yellow in superimposed image (right; magnification, x250). Most of the PrP signal in germinal centers and appeared to colocalize with the FDC network.
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Reconstituted mice were challenged intraperitoneally with scrapie prions. Surprisingly, all mice that received FLCs of either genotype, Prnp+/+ or Prnpo/o, from immunocompetent donors succumbed to scrapie after inoculation with a high dose of prions, and most mice succumbed after a low dose (Fig. 2). Transfer of FLCs from µMT donors, as well as omission of the adoptive transfer procedure, did not restore susceptibility to disease in any of the immune-deficient mice challenged with the low dose of prions. We also confirmed that by using high-dose inoculum, susceptibility to scrapie could be restored even in the absence of B cells and FDCs. However, reconstituted mice that received bone marrow from TCR–/– donors, which possess B cells and lack all T cells except those expressing TCR
receptors, regained susceptibility to scrapie, again confirming the dependency of infectibility on the presence of B cells (Fig. 2). By transmitting individual samples of brain and spleen from the scrapie-inoculated bone marrow chimeras, we observed restoration of infectious titers and PrPSc deposition in spleens and brains of recipient mice either carrying Prnp+/+ or Prnpo/o donor cells (9).
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FIGURE 2. Latency of scrapie in different bone marrow-reconstituted mice after intraperitoneal inoculation of RML, and average (–) of the incubation time. Transfer of Prnp+/+ or of Prnpo/o fetal liver cells (FLCs; closed triangles), but not of µMT FLCs (open circles), restored infectibility of immune-deficient mice on intraperitoneal inoculation with 3–4 logLD50 scrapie prions. An analogous trend was seen when 7–8 logLD50 were inoculated, although resistance to central nervous system disease of immunodeficient mice was often overcome by this high dose.
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Although B cells are clearly a cofactor in peripheral prion pathogenesis, the identity of those cells in which prions actually replicate within lymphatic organs is uncertain. In a further step to clarify this issue, we investigated whether spleen PrPSc was associated with FDCs in repopulated mice. Double-color immunofluorescence confocal microscopy revealed deposits of PrP-immunoreactive material in germinal centers, which appeared largely colocalized with the follicular dendritic network in spleens of reconstituted mice (Fig. 3).
Collectively, these findings are compatible with the hypothesis that cells whose maturation depends on B cells are responsible for accumulation of prions in lymphoid tissue such as the spleen. FDCs, although their origin remains rather obscure, are a likely candidate for the site of prion replication because their maturation correlates with the presence of B cells and their products. However, it is still possible that the follicular dendritic network serves merely as a reservoir for the accumulation of prions and that other B cell-dependent processes are involved in the transport of the infectious agent. Prions may be transported on or within B cells directly as they cross peripheral lymphoid tissue to localize in autonomic nerve terminals. Indeed, recent investigations have demonstrated that prion infectivity is mainly associated with B and T lymphocytes and less with a stromal fraction containing FDCs (12). Alternatively, antibodies or other B cell factors may bind prions and fulfill this role. This is particularly likely because PrPSc can be detected by immunohistochemistry in the germinal center area of lymphatic organs where immune complexes are deposited.
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Conclusion
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Peripheral prion pathogenesis, and ultimately neuroinvasion, is dependent on components of the host immune system. Collectively, these processes require either B cells per se or their products. At least one B cell-dependent event is the acquisition of a functional FDC network within the germinal centers of peripheral lymphoid tissue. These cells are the major sites of extraneuronal PrPC expression and probably the principal sites of PrPSc accumulation. The mechanism by which prions accumulate within lymphoid tissue remains to be established. An attractive hypothesis is that prions bind to antibodies that localize to the surface of FDC as a prion-antibody complex in a manner analogous to the normal function of FDC.
The second phase of neuroinvasion appears to be the progression of prions from lymphoid tissue to nerve endings of the sympathetic nervous system. This may occur by the direct transport of prions into peripheral lymphoid tissue or from reservoirs of infectivity associated with FDC, although no PrPSc has been detected in the autonomic peripheral nervous system so far. It is worthwhile noting that the innervation of lymphoid tissue is at least in part controlled by lymphocytes themselves, since both T and B cells secrete nerve growth factor and nerve terminals secrete a variety of factors to stimulate the immune system in kind (14). These factors may play a critical role in the neuroinvasion process and represent a critical site for modulation of disease progression. For example, drugs that act on lymphocytes or at the synaptic innervation of lymphoid tissue, or those that prevent cytokine release or block neurotransmission, may have a strong influence in the immune modulation and might represent useful tools for studying the cellular and molecular basis of prion neuroinvasion. A thorough understanding of the role of the immune system in peripheral prion pathogenesis is of immediate importance in assessing the risk of iatrogenic transmission of prions via exposure to blood or tissues from individuals suffering from preclinical prion disease.
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References
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Blättler T, Brandner S, Raeber AJ, Klein MA, Voigtländer T, Weissmann C, and Aguzzi A. PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain. Nature 389: 69–73, 1997.[Medline]
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Introduction
Peripheral route of infection
