Neuroimmune contacts between peripheral nervous system Neuroimmune contacts between peripheral nervous system and follicular dendritic cells in ovine lymph organs.and follicular dendritic cells in ovine lymph organs.

Toppets V1, Piret J1, Defaweux V2, Dorban G2, Jacqmot O3, Grobet L1, Antoine N1.

OBJECTIVEOBJECTIVEIn this preliminary approach, we examined the topography of the peripheral nervous system in ovine tonsils and in their draining lymph nodes, first potent target sites for the disease-causing agents replication.

MATERIAL AND METHODSMATERIAL AND METHODS

Palatine, pharyngeal tonsils and medial retropharyngeal lymph nodes were removed from sheep 6 to 8 month old and cryosections were processed for immunofluorescence. FDCs were stained with FDC-B1 antibody. Nerve fiberswere identified with specific polyclonal antibodies directed against intermediate neurofilaments L, M and H. All samples were observed with a Leica confocal microscope.

BACKGROUNDBACKGROUNDScrapie is a lethal disease affecting sheep and goats. In sheep, the PrP genotype is known to modulate susceptibility/ resistance to TSE. The causative agent, PrPSc, enters the organism mainly by the oral route and rapidly invades the mucosal associated lymphoid tissues. It is now clearly established that follicular dendritic cells (FDCs), stromal-differentiated cells present in the germinal centre of the lymph follicles are greatly implicated in the retention and replication of PrPSc after scrapie infection. Nevertheless, how the TSE agents initially spread from the FDCs to the peripheral nervous system before reaching the central nervous system is not known.

Lymphoid Follicles

FDCsPeripheral nerves fibresPeripheral nerves fibres

Central Nervous System

?

Primary Antibodies Target Secondary Antibodies

FDC-B1

mouse anti-bovineFDCs

Alexa 546

goat anti-mouse

Anti-Neurofilaments H (1)

Neurofilaments H

Alexa 488

goat anti-rabbit

Anti-Neurofilaments L (2)

Neurofilaments L

Anti-Neurofilaments M(3)

(1),(2) et (3) rabbit anti-mouse

Neurofilaments M

Table 1: primay and secondary antibodies used

RESULTSRESULTS

FDC-B1 immunostaining localized the FDCs network in the light zone of the germinal centre follicles. In all lymph organs, the neurofilaments M were the most abundant in the follicular area.They were preferentially located in the mantle zone surrounding the FDC network and, as illustrated, some nerves fibres propagated through the geminal centre. This pattern of innervation was particularly developed in the palatine tonsils.

CONCLUSIONCONCLUSION

Our preliminary results pointed an hypothetical transfer of prion protein directly from the germinal centre, to the peripheral nervous system via neurofilaments M endings in contact with follicular dendritic cells.

Ovine palatine tonsil : FDCs network in red - neurofilaments M in green. Gx400.

Neurofilaments M :Graph 1

Graph 2

Some proven contacts (yellow fluorescence points) between FDCs and neurofilaments M were highlighted by a combined approach of image and spectral analyses.

Neurofilaments H and L:

In tonsils, the rare nervous fibres H or L were mostly localized in the sub-epithelial ( ou lamina propria?) connective tissues. In lymph nodes, they were detected in the capsule and in the trabeculae. In both cases, the neurofilaments H and L were distant from the lymphoid follicles and consequently, no contact have been detected.

1. Department of Morphology and Pathology (DMP), Laboratory of Animal Histology and Embryology, Faculty of Veterinary Medicine (FMV), University ofLiege (Ulg), Belgium; 2. Laboratory of Human Histology, Faculty of Medicine, ULg, Belgium; 3. DMP, Laboratory of Animal Anatomy, FMV, ULg,,

Belgium.