Scrapie affects the maturation cycle and immune complex trapping by follicular dendritic cells in mice

Abstract

Transmissible spongiform encephalopathies (TSEs) or prion diseases are infectious neurological disorders of man and animals, characterised by abnormal disease-associated prion protein (PrP(d)) accumulations in the brain and lymphoreticular system (LRS). Prior to neuroinvasion, TSE agents often accumulate to high levels within the LRS, apparently without affecting immune function. However, our analysis of scrapie-affected sheep shows that PrP(d) accumulations within the LRS are associated with morphological changes to follicular dendritic cells (FDCs) and tingible body macrophages (TBMs). Here we examined FDCs and TBMs in the mesenteric lymph nodes (MLNs) of scrapie-affected mice by light and electron microscopy. In MLNs from uninfected mice, FDCs could be morphologically categorised into immature, mature and regressing forms. However, in scrapie-affected MLNs this maturation cycle was adversely affected. FDCs characteristically trap and retain immune complexes on their surfaces, which they display to B-lymphocytes. In scrapie-affected MLNs, some FDCs were found where areas of normal and abnormal immune complex retention occurred side by side. The latter co-localised with PrP(d) plasmalemmal accumulations. Our data suggest this previously unrecognised morphology represents the initial stage of an abnormal FDC maturation cycle. Alterations to the FDCs included PrP(d) accumulation, abnormal cell membrane ubiquitin and excess immunoglobulin accumulation. Regressing FDCs, in contrast, appeared to lose their membrane-attached PrP(d). Together, these data suggest that TSE infection adversely affects the maturation and regression cycle of FDCs, and that PrP(d) accumulation is causally linked to the abnormal pathology observed. We therefore support the hypothesis that TSEs cause an abnormality in immune function.

Figure 1. Detection of PrP^d, IgG, IgM and ubiquitin in MLNs from uninfected and scrapie-affected mice.

Bars = 35 µm. (A to D) Resin-embedded MLN from an uninfected mouse, (1 µm serial sections). (A) No PrP^d immunolabelling detected in MLNs from uninfected mice. (B) Thin branches of IgG immunolabelling are interspersed between lymphocytes of the secondary follicles. (C) IgM immunolabelling is similar to that of IgG but appeared less intense. (D) Diffuse ubiquitin labelling is abundant throughout the follicle, closely surrounding the nuclei of many cells. (E to H) Resin-embedded MLN from a scrapie-affected mouse (1 µm serial sections). (E) Linear FDC PrP^d immunolabelling is primarily present within the light zone (LZ) of the follicle interspersed between lymphocytes. Intense puncta which correspond with intracytoplasmic TBM labelling, are present within both the light and dark zones (DZ) of the follicle (and insert). Darkly stained apoptotic cells, or tingible bodies, are also present within the TBM cytoplasm (insert). (F) Intense IgG labelling corresponds with the FDC pattern of labelling seen in panel E. Accumulations appear more expanded within the extracellular space when compared with IgG labelling of the uninfected control shown in panel B. (G) Patterns of IgM immunolabelling are similar to those observed in both panels E and F, however labelling is less intense. (H) Patterns of ubiquitin immunolabelling are similar in distribution to those in normal animals (D), however, the magnitude appeared considerably greater.

Figure 2. FDC morphology in uninfected mice.

Uranyl acetate/lead citrate stain. (A) Immature FDC. The nucleus has a thin border of euchromatin and is surrounded by limited cytoplasm containing few organelles. Dendrites are sparse and where present do not accumulate electron dense deposit within the extracellular space (insert and arrow). Bar = 1 µm. (B) Mature FDC. Dendrites are more developed and form small knots, between which curvi-linear electron dense deposit of uniform thickness can be seen (insert and arrow). Bar = 1 µm. (C) Regressing FDC. Dendrites appear distinct, with tissue spaces appearing between processes. The uniform electron dense extracellular deposit surrounding dendrites is lacking (arrows). Bar = 2 µm. (D). TBM. Cytoplasm contains apoptotic bodies (asterisks), in addition to endososomes and lysosomes. Bar = 1 µm.

Figure 3. FDC morphology and sites of PrP^d accumulation in scrapie-affected MLNs. PrP^d immunogold labelling.

(A) Early mature FDC. PrP^d labelling is limited to the plasmalemma and adjacent extracellular space of FDC dendritic processes. An electron dense line intermediate between dendrites can be seen and is indicative of immune complex retention (arrows). No PrP^d is associated with the immune complex material. Where PrP^d accumulates, no linear dense line (presumptive immune complex) is retained (arrowhead and insert). Dendritic folding is associated with both PrP^d accumulation on the plasmalemma and the loss of the normal linear immune complex retention (insert). Bar = 1 µm. (B) Mature FDC. Lymphocyte (ly) emperipolesed by PrP^d-expressing FDC processes. Dendrites are convoluted and form intricately interwoven complexes. The space between dendrites is expanded and contains abundant electron dense deposit (arrows) which is predominantly unlabelled for PrP^d. Bar = 2 µm. (C) Mature FDC. PrP^d accumulates on the plasmalemma of FDC dendrites. Extracellular electron dense deposit is not abundant. Infrequent indistinct spherical or ovoid exosome-like structures are present in close proximity to the plasmalemma of the FDC (arrows and insert). Bar = 0.5 µm. (D) Regressing FDC. The cytoplasm of the regressing FDC (asterisk) is more electron dense than adjacent cells. Dendrites form distinct rod-like projections, or short thick structures. PrP^d is primarily limited to the plasmalemma of the dendrites closer to the cell body, however limited PrP^d is also associated with the rod-like dendrites further from the cell body, albeit at a considerably lower level (arrow). A lymphocyte lies adjacent to the FDC (ly). Bar = 1 µm.

Figure 4. IgG immunogold labelling upon FDCs in MLNs from uninfected and scrapie-affected mice.

(A) FDC processes from an uninfected mouse. Immunogold labelling is closely associated with the electron dense deposit between dendritic processes of a mature FDC. At this magnification, dense lines (the intermediate dense line - arrows) are just visible within the dense deposit. The dotted line of the insert highlights this intermediate dense line. Bar = 0.2 µm. (B) Early stage mature FDC from a scrapie-affected mouse. Although cell dendrites are extended, the space between opposing dendrites remains uniform. IgG immunogold labelling is restricted to the electron dense deposit held in the intermediate space between profiles. Bar = 1 µm. (C) Mature FDC from a scrapie-affected mouse. Extensive immunogold labelling is associated with the extensive electron dense deposit between processes of this scrapie-specific mature form of FDC. No intermediate dense line is visible in this irregular electron dense deposit Bar = 1 µm. (D) Mature FDC from a scrapie-affected mouse. Electron dense material is not abundant between opposing dendrites and IgG labelling is sparse. Where present, immunogold labelling is clearly limited to areas adjacent to dendrites. Bar = 1 µm.

Figure 5. Ubiquitin is abundant within the follicles of MLNs from scrapie-affected mice.

(A) Ubiquitin immunogold labelling is clearly limited to the plasmalemma of a mature FDC from a scrapie-affected mouse. Bar = 1 µm. (B) Abundant ubiquitin is present within endo/lysosomal structures within the cytoplasm of a TBM. Most immunogold labelling is detected within endosomes (arrow). Lysosomes in contrast have a limiting membrane (arrowhead) and contain little ubiquitin. Bar = 0.5 µm.

Figure 6. Diagram showing the maturation cycles of normal and scrapie-affected FDCs.

In unifected mice, following Ag-stimulation, immature FDCs (A) develop and trap immune complex within the extracellular space between opposing FDC dendrites (B). Finally, FDCs loose their capacity to trap immune complex and regress (C). In contrast, in scrapie-affected animals, mature FDCs develop to form abnormal disease-specific forms of the cell, initially with accumulation of PrP^d on the plasmalemma (D), followed by excess putative immune complex and abnormal extension of dendrites (E), and immune complex and sparse exosomes (F). In the current study our data suggest that the final stage of the FDC maturation cycle in scrapie-affected animals leads to the loss of PrP^d from the plasmalemma as the FDCs and secondary follicles regress (G). In all diagrams, purple indicates putative immune complex held between dendrites, exosome-like structures are highlighted in yellow (F). Red arrows indicate scrapie-specific stages of maturation while dark blue arrows highlight normal stages. The PrP^d molecule is coloured red and dendrites are shaded in grey.