Abstract
The role of B cells in multiple sclerosis (MS) is increasingly recognized. B cells undergo compartmentalized redistribution in blood and cerebrospinal fluid (CSF) during active MS, whereby memory B cells accumulate in the CSF. While B-cell trafficking across the blood–brain barrier has been intensely investigated, cellular diapedesis through the blood–CSF barrier (BCSFB) is incompletely understood. To investigate how B cells interact with the choroid plexus to transmigrate into the CSF we isolated circulating B cells from healthy donors (HC) and MS patients, utilized an inverted cell culture filter system of human choroid plexus papilloma (HIBCPP) cells to determine transmigration rates of B-cell subsets, immunofluorescence, and electron microscopy to analyze migration routes, and qRT-PCR to determine cytokines/chemokines mediating B-cell diapedesis. We also screened the transcriptome of intrathecal B cells from MS patients. We found, that spontaneous transmigration of HC- and MS-derived B cells was scant, yet increased significantly in response to B-cell specific chemokines CXCL-12/CXCL-13, was further boosted upon pre-activation and occurred via paracellular and transcellular pathways. Migrating cells exhibited upregulation of several genes involved in B-cell activation/migration and enhanced expression of chemokine receptors CXCR4/CXCR5, and were predominantly of isotype class switched memory phenotype. This antigen-experienced migratory subset displayed more pronounced chemotactic activities in MS than in HC and was retrieved in intrathecal B cells from patients with active MS. Trafficking of class-switched memory B cells was downscaled in a small cohort of natalizumab-exposed MS patients and the proportions of these phenotypes were reduced in peripheral blood yet were enriched intrathecally in patients who experienced recurrence of disease activity after withdrawal of natalizumab. Our findings highlight the relevance of the BCSFB as important gate for the entry of potentially harmful activated B cells into the CSF.
Highlights
The central nervous system (CNS) is separated from bloodderived leukocytes and soluble factors by complex barrier systems, among them the blood–brain barrier (BBB), which encompasses deep parenchymal microvessels; the blood– meningeal barrier (BMB); and the blood–cerebrospinal fluid (CSF) barrier (BCSFB), which encases the choroid plexus, epithelium-based structures localized within the brain ventricles that produce CSF [1,2,3,4]
TM transwell filters were fixed in 2.5% glutaraldehyde, postfixed with 1% osmium tetroxide, cut into strips, dehydrated, and embedded in epoxy resin. 60 nm ultrathin sections were cut with an ultramicrotome, contrasted with uranyl acetate/lead citrate, and analyzed with a transmission electron microscopy (TEM) JEM140 equipped with a digital camera (TVIPS F420)
The BSCFB, together with the BBB and the BMB, tightly controls the influx of cells and macromolecules into the CNS and, when dysfunctional, might promote and sustain chronic inflammation such as occurs in multiple sclerosis (MS), a prototypical autoimmune disorder of the CNS. This may be true for B cells, which are virtually absent in normal CSF, yet in patients with MS accumulate predominantly as classswitched memory (CSM)
Summary
The central nervous system (CNS) is separated from bloodderived leukocytes and soluble factors by complex barrier systems, among them the blood–brain barrier (BBB), which encompasses deep parenchymal microvessels; the blood– meningeal barrier (BMB); and the blood–cerebrospinal fluid (CSF) barrier (BCSFB), which encases the choroid plexus, epithelium-based structures localized within the brain ventricles that produce CSF [1,2,3,4]. While barely detectable in normal CSF, B-lineage cells are clonally expanded in CSF and brain tissues from MS patients [17,18,19,20] This process appears to be driven by a compartmentalized redirection of memory B-lineage cells from blood into CSF, which paves the way for local conversion into antibody-secreting effector cells, as we have shown in a previous study [21]. These findings strongly emphasize a role of the BCSFB in initiating and perpetuating aberrant CNS B-cell-mediated immune responses in MS. We aimed to decipher the key B-cell subset involved in shifting between the systemic and intrathecal compartments
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