Accumulation profiles of PrP(Sc) in hemal nodes of naturally and experimentally scrapie-infected sheep

Abstract

Background: In classical scrapie, the disease-associated abnormal isoform (PrP(Sc)) of normal prion protein accumulates principally in the nervous system and lymphoid tissues of small ruminants. Lymph nodes traffic leukocytes via lymphatic and blood vasculatures but hemal nodes lack lymphatic vessels and thus traffic leukocytes only via the blood. Although PrP(Sc) accumulation profiles are well-characterized in ovine lymphoid tissues, there is limited information on such profiles in hemal nodes. Therefore, the objective of this study was to compare the follicular accumulation of PrP(Sc) within hemal nodes and lymph nodes by prion epitope mapping and western blot studies.

Results: Our studies found that PrP(Sc) accumulation in 82% of animals' abdominal hemal nodes when PrP(Sc) is detected in both mesenteric and retropharyngeal lymph nodes collected from preclinical and clinical, naturally and experimentally (blood transfusion) scrapie-infected sheep representing all three major scrapie-susceptible Prnp genotypes. Abdominal hemal nodes and retropharyngeal lymph nodes were then used to analyze immune cell phenotypes and PrP(Sc) epitope mapping by immunohistochemistry and PrP(Sc) banding patterns by western blot. Similar patterns of PrP(Sc) accumulation were detected within the secondary follicles of hemal nodes and retropharyngeal lymph nodes, where cellular labeling was mostly associated with macrophages and follicular dendritic cells. The pattern of PrP(Sc) accumulation within hemal nodes and retropharyngeal lymph nodes also did not differ with respect to epitope mapping with seven mAbs (N-terminus, n = 4; globular domain, n = 2; C-terminus, n = 1) in all three Prnp genotypes. Western blot analysis of hemal node and retropharyngeal lymph node homogenates revealed identical three banding patterns of proteinase K resistant PrP(Sc).

Conclusion: Despite the anatomical difference in leukocyte trafficking between lymph nodes and hemal nodes, the follicles of hemal nodes appear to process PrP(Sc) similarly to lymph nodes.

Figure 1

Immunolabeling of PrP^Sc in the hemal nodes from a scrapie-infected sheep. PrP^Sc immunolabeling (dark red) was visible in the secondary follicles of abdominal hemal nodes (animal ID 3777) when labeled with anti-prion mAbs 12B2 (A), P4 (B), 1E4 (C), 8G8 (D), L42 (E), SAF84 (F), F99.97.6.1 (G) and retropharyngeal lymph nodes labeled with SAF84 (I). The FDC-type of PrP^Sc immunolabeling was visible in the hemal nodes of this animal with 12B2 and SAF84 mAbs. PrP^Sc immunolabeling was not observed in the hemal nodes’ secondary follicles when anti-prion mAbs were replaced with isotype-matched control immunoglobulins (H; same tissue block shown in A-G). Scale bar = 50 μm.

Figure 2

Immunolabeling of PrP^Sc in the retropharyngeal lymph nodes from a scrapie-infected sheep. The FDC-type of PrP^Sc immunolabeling (dark red) was visible in the light zones of secondary follicles in the retropharyngeal lymph nodes (animal ID 3753) when labeled with all seven anti-prion mAbs; 12B2 (A), P4 (B), 1E4 (C), 8G8 (D), L42 (E), SAF84 (F) and F99.97.6.1 (G). However, such FDC-type of PrP^Sc labeling was not observed in this animal when paired hemal nodes were labeled with any of these antibodies. [Example; F99/97.6.1 (H) and SAF84 (I)]. Scale bar = 50 μm.

Figure 3

Double immunolabeling of leukocyte subsets and PrP^Sc in the hemal nodes and retropharyngeal lymph nodes from scrapie-infected sheep. Retropharyngeal lymph nodes (A, C, E, G) and abdominal hemal nodes (B, D, F, H) were immunolabeled with T lymphocyte-specific (CD3; animal ID 4496; A and B), B lymphocyte-specific (CD79a; animal ID 4496; C and D), macrophage-specific (CD163; animal ID 4489; E and F) and follicular dendritic cell-specific (120 kDa antigen; animal ID 3776; G and H) Abs along with anti-prion F99/97.6.1 mAb. Under these double labeling conditions, PrP^Sc appears in red while each cell-type appears in brown. Enlarged pictures of co-labeled cells with cell-type specific mAb and PrP^Sc are inserted into the right lower corner of each panel. Scale bar = 50 μm.

Figure 4

Detection of characteristic proteinase K resistant three isoforms of PrP^Sc from hemal nodes and retropharyngeal lymph nodes by western blot assays. Western blot analysis was performed with retropharyngeal lymph node (A) and abdominal hemal node (B) homogenates prepared from experimentally and naturally scrapie-infected sheep. Lanes 1 – 6, experimentally (blood transfusion recipient) scrapie-infected sheep (ID 4489, 4493, 4494, 4495, 4496, and 4498 respectively); lanes 7 -9 naturally scrapie-infected sheep (ID 3349, 3779 and 3781 respectively). Lane 10, scrapie uninfected control sheep (ID 4492). To concentrate PrP^Sc, proteinase K treated samples were incubated with sodium phosphotungstic acid and PrP^Sc was detected using F99/97.6.1 mAb. The positions of the molecular mass markers (in kDa) are shown to the left of the blots.