Is peripheral immunity regulated by blood-brain barrier permeability changes?

S100B is a reporter of blood-brain barrier (BBB) integrity which appears in blood when the BBB is breached. Circulating S100B derives from either extracranial sources or release into circulation by normal fluctuations in BBB integrity or pathologic BBB disruption (BBBD). Elevated S100B matches the clinical presence of indices of BBBD (gadolinium enhancement or albumin coefficient). After repeated sub-concussive episodes, serum S100B triggers an antigen-driven production of anti-S100B autoantibodies. We tested the hypothesis that the presence of S100B in extracranial tissue is due to peripheral cellular uptake of serum S100B by antigen presenting cells, which may induce the production of auto antibodies against S100B. To test this hypothesis, we used animal models of seizures, enrolled patients undergoing repeated BBBD, and collected serum samples from epileptic patients. We employed a broad array of techniques, including immunohistochemistry, RNA analysis, tracer injection and serum analysis. mRNA for S100B was segregated to barrier organs (testis, kidney and brain) but S100B protein was detected in immunocompetent cells in spleen, thymus and lymph nodes, in resident immune cells (Langerhans, satellite cells in heart muscle, etc.) and BBB endothelium. Uptake of labeled S100B by rat spleen CD4+ or CD8+ and CD86+ dendritic cells was exacerbated by pilocarpine-induced status epilepticus which is accompanied by BBBD. Clinical seizures were preceded by a surge of serum S100B. In patients undergoing repeated therapeutic BBBD, an autoimmune response against S100B was measured. In addition to its role in the central nervous system and its diagnostic value as a BBBD reporter, S100B may integrate blood-brain barrier disruption to the control of systemic immunity by a mechanism involving the activation of immune cells. We propose a scenario where extravasated S100B may trigger a pathologic autoimmune reaction linking systemic and CNS immune responses

Summary of experimental results testing for the presence of S100B at the protein, mRNA levels or after injection of labeled S100B.

<p>+ indicates presence, ++ presence at levels significantly greater than in tissues labeled with +. ND =  Not determined, - indicates absence of measurable signal.</p

Endothelial cells take up circulating but not CNS-derived S100B.

<p>(<b>A</b>) Lack of significant endogenous immunoreactivity for S100B in BBB endothelial cells. The <i>arrows</i> point to faintly stained capillaries in the hippocampus (CA1 region). This staining was accounted for by glial end feet positive for S100B. Note the endogenous S100B immunostaining of astrocytes located within the pyramidal cell layer. (<b>B</b>) Uptake of circulating (exogenous) S100B by endothelial cells after injection of green S100B is observed in most systemic vessels. The example shows the appearance of an arteriole and capillaries in a lymph node, indicated by <i>arrowheads</i>. (<b>C1</b>) and (<b>C2</b>) show the uptake of exogenous, circulating S100B by endothelial cells of the retinal barrier. Note the uptake indicated by <i>arrows</i> and the various retinal layers. (<b>D1-3</b>) Co-localization of CD31 and exogenous S100B indicates uptake by endothelial cells. Endothelial cells take up S100B after systemic injection of labeled protein. The results shown here use an immunohistochemical validation by an endothelium-specific marker to corroborate the results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101477#pone-0101477-g001" target="_blank">Figure 1</a>. In fact, the cells demonstrating S100B uptake (<i>green</i>) were also positive for the endothelial marker CD31. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101477#pone.0101477.s003" target="_blank">Figure S3</a> A for CNS BBB endothelial cells. <i>G =  ganglion cell layer, IN  =  inner nuclear layer, ON  =  outer nuclear layer, RC  =  rods and cones, C =  choroid, S =  sclera</i>. AF 488-tagged S100B was injected to achieve a serum concentration of 0.12 ng/mL to mimic blood-brain barrier disruption (ref. 13).</p

Schematic representation of the events that follow BBBD.

<p>See text for details.</p

Circulating S100B fails to invade barrier organs; however S100B gene transcription and protein synthesis occurs in both brain and testis.

<p>Injection (exogenous) strategies demonstrate privilege of barrier organs to transendothelial diffusion of S100B while immunodetection of endogenous S100B demonstrates brain- and testis specific S100B protein by astrocytes and Sertoli cells. mRNA detection in the same barrier organs confirms S100B expression. <b>(A1)</b> shows the lack of fluorescent signal (Alexa Fluor (AF) 488, in <i>green</i>) in brain regions where endogenous S100B was readily detected <b>(A2)</b>. The section used for immunohistochemistry contained the CA2 sector of the hippocampus. Note that, as expected, S100B was present in glial cells but not in neurons; neuronal cell bodies in hippocampal CA2 region are seen as unstained ghosts. Testicular tissue yielded similar results albeit in testis the barrier is established by Sertoli and not endothelial cells <b>(B1 and B2)</b>. Note that intravascular S100B was restricted to the stroma of the seminiferous tubules in the testis where endogenous S100B was not present. S100B+ cells in the stroma (<i>arrowheads</i>) are CD4+ dendritic cells (<i>insert</i> in <b>B2</b>). DAPI (<i>blue</i>) was added as a nuclear stain <b>(B1)</b>. AlexaFlour 488-tagged S100B was injected to mimic blood-brain barrier disruption (e.g., ref. 13). The labeled protein was allowed to circulate 3 hrs. prior to tissue harvesting. The mRNA bands shown reflect levels of expression by brain and testis. <i>Sem. T.  =  seminiferous tubule</i>.</p

Circulating S100B is taken up by peripheral immune cells.

<p>AlexaFlour 488-tagged S100B protein accumulated in Langerhans cells of the skin (<b>A1, 2</b>), thymic dendritic cells (<b>B1, 2</b>), and in dendritic cells in lymph nodes (a para-aortic lymph node is shown in <b>C1, 2</b>). Note the lack of uptake by non-immune surrounding tissue. Also note the typical appearance of dendritic cells in nodal tissues. Injection consisted of AlexaFlour 488-tagged S100B at a concentration of 0.12 ng/mL introduced via tail vein and allowed to circulate 2–3 hrs. prior to tissue harvesting; DAPI (<i>blue</i>) was added as a nuclear stain. Note the different magnifications among the panels, with scale bar  = 200 µm in B1, 100 µm in A2 and 50 µm in all other panels. Rats were injected with a combination of AlexaFlour 594-tagged S100A1 (<i>red</i>) and AlexaFlour 488-tagged S100B (<i>green</i>). Skin (<b>D</b>) and splenic (<b>E</b>) tissue revealed that Langerhans cells (<i>arrows</i> in D) and splenic cells within the germinal center (<i>GC</i>), mantel zone (<i>MnZ</i>) and marginal zone (<i>MaZ</i>) demonstrate uptake of both S100A1 and S100B; however, the extent and intensity of S100B uptake was much greater as also evident in the merged figures (D3, E3). <b>F</b> shows the preferential segregation of S100B at the membrane of dendritic cells in skin (<b>F1</b> and <b>F2</b>) and thymus (<b>F3</b>); greater detail of a Langerhans cell from D2 (box) is shown in F1 in order to highlight membrane staining. 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; DAPI (blue) was added as a nuclear stain. <i>GC  =  germinal center, MnZ  =  mantle zone, MaZ  =  marginal zone</i>.</p

Splenic S100B-positive cells change in both number and morphology following pilocarpine-induced seizure.

<p>S100B positive cells can be viewed within splenic follicles both in an unmanipulated animal (<b>A</b>), as well as following simulation of BBBD via IV injection of Alexa Fluor 488-labeled S100B (<b>B</b>) and BBBD from pilocarpine administration (<b>C</b>). 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 (<b>C</b>), 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 (<b>D1–D2</b>); 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 <b>A</b> and <b>C</b> were treated with mouse anti-S100B antibody (Ab) and donkey anti-mouse 2° Ab conjugated to FITC (Jackson). In <b>D1–D2</b>, 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 <b>E–F</b>, sections shows rats injected with S100B tracer in control (<b>E1–E3</b>) and pilocarpine administrated rats (<b>F1–F3</b>). Co-localization of S100B+CD86 (F1) and high individual staining of CD86+ (<b>F2</b>) in pilocarpine compared to controls <b>E1</b> and <b>E2</b> indicates that activated immune cells capture S100B. The dendritic cell nature of these cells was further demonstrated by their CD86+ staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101477#pone.0101477.s003" target="_blank">Figure S3</a>). Scale bar in A = 100 µm for all images. <i>GC  =  germinal center, MnZ  =  mantle zone, MaZ  =  marginal zone, WP  =  white pulp, RP  =  red pulp</i>.</p

Comparison of S100B and S100A1 sequences to underscore similarities (normal character) and differences (in bold).

<p>There is 94% (4 aa are different) similarity between bovine S100B used for these experiments and rat S100B. Thus, the uptake by immune cells is not due to foreign antigen sequence. Same considerations can be made for human S100A1 used as a tracer, since the human protein used shared 96% identity with rat protein.</p