Murine Scrapie Infection Causes an Abnormal Germinal Centre Reaction in the Spleen

Abstract

Follicular dendritic cells (FDCs) of the lymphoreticular system play a role in the peripheral replication of prion proteins in some transmissible spongiform encephalopathies (TSEs), including experimental murine scrapie models. Disease-specific PrP (PrP^d) accumulation occurs in association with the plasmalemma and extracellular space around FDC dendrites, but no specific immunological response has yet been reported in animals affected by TSEs. In the present study, morphology (light microscopical and ultrastructural) of secondary lymphoid follicles of the spleen were examined in mice infected with the ME7 strain of scrapie and in uninfected control mice, with or without immunological stimulation with sheep red blood cells (SRBCs), at 70 days post-inoculation or at the terminal stage of disease (268 days). Scrapie infection was associated with hypertrophy of FDC dendrites, increased retention of electron-dense material at the FDC plasma membrane, and increased maturation and numbers of B lymphocytes within secondary follicles. FDC hypertrophy was particularly conspicuous in immune-stimulated ME7-infected mice. The electron-dense material was associated with PrP^d accumulation, as determined by immunogold labelling. We hypothesize that immune system changes are associated with increased immune complex trapping by hypertrophic FDCs expressing PrP^d molecules at the plasmalemma of dendrites, and that this process is exaggerated by immune system stimulation. Contrary to previous dogma, these results show that a pathological response within the immune system follows scrapie infection.

Introduction

Transmissible spongiform encephalopathies (TSEs) are a family of slowly progressive neurodegenerative disorders, consisting of infectious, familial and sporadic forms of disease in both animals and man. The TSEs include bovine spongiform encephalopathy (BSE) of cattle and exotic ungulates, scrapie of sheep and goats, and Creutzfeldt-Jakob disease (CJD), kuru and Gertsmann-Straüssler syndrome (GSS) of human beings. TSEs can be naturally, experimentally or iatrogenically transmitted to several mammalian species and are characterized by the accumulation of an abnormal disease-specific isoform of a host-encoded cell surface glycoprotein called prion protein (PrP^d). The normal PrP molecule (PrP^c) is expressed abundantly in the central nervous system (CNS) (Manson et al., 1992, Ford et al., 2002) and less abundantly in many other tissues (Oesch et al., 1985), while PrP^d accumulates in the CNS in many of the TSEs and also in the peripheral nervous system and lymphoreticular tissues in most experimental animal models.

The role of the lymphoreticular system (LRS) in the pathogenesis of TSEs has been extensively studied (Fraser and Dickinson, 1970, Mabbott and Bruce, 2001). Initial investigations into which cells within the LRS permit prion replication demonstrated that T-cell depletion by adult or neonatal thymectomy had no effect on scrapie pathogenesis, suggesting that T cells were not critical for scrapie pathogenesis. However, surgical or genetic splenectomy prolonged the incubation period in several scrapie models (Kimberlin and Walker, 1989), demonstrating the pathogenetic importance of the spleen and its cellular components. Further studies revealed that exposure of scrapie-infected mice to lethal doses of gamma irradiation had no effect on pathogenesis, or on prion replication in the spleen, suggesting that a long-lived radiation-resistant cell was important for neuroinvasion and replication of the infective agent (Fraser and Dickinson, 1978, Fraser and Farquhar, 1987).

Genetically immunodeficient (SCID) mice lack functional B and T lymphocytes, and follicular dendritic cells (FDCs) in such mice remain immature due to lack of stimulation from B cells (Bosma et al., 1983, Kapasi et al., 1993). SCID mice are also relatively resistant to peripheral (i.e., non-CNS) inoculation with scrapie (Kitamoto et al., 1991, O'Rourke et al., 1994, Brown et al., 1997). The lymphocyte population in SCID mice can be restored with bone marrow from immunocompetent donors, enabling FDCs to mature (Kapasi et al., 1993). Lymphocyte-reconstituted SCID mice infected peripherally with scrapie develop the disease after incubation periods comparable with those of immunocompetent mice, confirming that cells of the immune system play a significant role in the peripheral pathogenesis of the disease (Fraser et al., 1996, Lasmezas et al., 1996).

While studies with SCID mice produced strong evidence in favour of a critical role for FDCs in pathogenesis, the possible role of lymphoid cells could not be excluded. A more conclusive role for FDCs in scrapie replication was provided by studies in chimaeric mice, in which PrP was expressed on FDCs but not on lymphocytes, and vice versa. In these models, replication of infectivity and progression of infection to the CNS depended upon the presence of mature PrP-expressing FDCs, and were independent of the expression of PrP on lymphocytes (Brown et al., 1999, Brown et al., 2000).

FDCs are accessory cells that occur only in lymphoid follicles, where they are tightly surrounded by lymphocytes (Tew et al., 1982). During germinal centre reactions, the function and morphology of FDCs change markedly. On stimulation, FDC processes elongate and make contact with numerous lymphocytes, while their surface receptors allow them to trap immune complexes which can be retained for extended periods. The immune complexes are then presented to, and processed by, B lymphocytes. Within the spleen, FDCs, unstimulated B cells and some macrophage populations are contained in the primary follicle of the white pulp region. This region is characterized by closely packed lymphocytes and is divided into two zones, namely, the follicle itself and the surrounding periarteriolar lymphoid sheath (PALS), which contains the arteriole, T cells and macrophages. The marginal zone tightly surrounds the PALS and merges into the highly vascularized red pulp.

In previous morphological studies of spleens from mice terminally affected by scrapie, FDCs formed highly convoluted labyrinthine structures, with irregular, often abundant, electron-dense deposits associated with cell dendrites (Jeffrey et al., 2000). These deposits occasionally contained PrP^d -associated amyloid fibrils, which were seen to a lesser extent in tissues obtained from mice at pre-clinical stages of disease. Immunogold labelling was associated with plasmablast emperipolesis by FDC dendrites (i.e., FDC dendrite-encircled plasmablasts), and with the extracellular space surrounding FDC dendritic processes. Coated pits were more abundant on the surface of FDCs from scrapie-infected animals than from normal control animals; PrP^d labelling, however, was not associated with these structures. In the spleens of pre-clinically and terminally affected animals, immunogold labelling was also associated with tingible body macrophage (TBM) lysosomes. Immunolabelling within these lysosomal compartments is predominantly associated with the more electron-dense areas. This lysosomal PrP^d is probably scavenged from the extracellular space or from FDC processes (Jeffrey et al., 2000). In sheep TBMs, PrP^d lacks the N terminus of the PrP, which further supports the suggestion that TBMs internalize and digest extra-cellular PrP (Jeffrey et al., 2000). The aims of the present study were (1) to characterize the morphological response of the secondary follicle to scrapie infection, and (2) to determine whether this response was normal as compared with other forms of exogenous antigenic stimulation.