Is Peripheral Immunity Regulated by Blood-Brain Barrier Permeability Changes?
Figure 5
Splenic S100B-positive cells change in both number and morphology following pilocarpine-induced seizure.
S100B positive cells can be viewed within splenic follicles both in an unmanipulated animal (A), as well as following simulation of BBBD via IV injection of Alexa Fluor 488-labeled S100B (B) and BBBD from pilocarpine administration (C). The pattern of staining is preserved in all 3 conditions, however, staining is clearly augmented in BBBD simulation and to a much greater degree in actual induced BBBD. S100B-labeled lymphocytes and dendritic cells can be observed in all regions of the splenic follicle. Note that the morphology of labeled cells also appears to change with induction of BBBD in (C), where cells appeared to have a more dendritic and interconnected staining pattern than in other conditions. The identity of these cells was verified via the immune cell markers, CD4 and CD8 immunostaining (D1–D2); S100B+ and CD4+/CD8+ double positive cells are again found throughout the follicle, with emphasized staining in the marginal zone vs other areas. Injection consisted of AlexaFlour 488-tagged S100B at a concentration of 0.10–0.12 ng/mL introduced via tail vein and allowed to circulate 2–3 hrs prior to tissue harvesting. For induced BBBD, the animal was treated with pilocarpine and spleen was removed prior to onset of status epilepticus. Sections in A and C were treated with mouse anti-S100B antibody (Ab) and donkey anti-mouse 2° Ab conjugated to FITC (Jackson). In D1–D2, sections were treated with rabbit anti-S100B Ab and donkey rabbit 2° Ab conjugated to Texas Red (Jackson) and rat anti-CD4 or CD8 antibody and mouse anti-rat 2° Ab conjugated to FITC (Jackson). DAPI was added as a nuclear stain. In E–F, sections shows rats injected with S100B tracer in control (E1–E3) and pilocarpine administrated rats (F1–F3). Co-localization of S100B+CD86 (F1) and high individual staining of CD86+ (F2) in pilocarpine compared to controls E1 and E2 indicates that activated immune cells capture S100B. The dendritic cell nature of these cells was further demonstrated by their CD86+ staining (Figure S3). Scale bar in A = 100 µm for all images. GC = germinal center, MnZ = mantle zone, MaZ = marginal zone, WP = white pulp, RP = red pulp.