Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Nov 16;13(1):292.
doi: 10.1186/s12974-016-0755-8.

Appearance of claudin-5 + Leukocytes in the Central Nervous System During Neuroinflammation: A Novel Role for Endothelial-Derived Extracellular Vesicles

Affiliations
Free PMC article

Appearance of claudin-5 + Leukocytes in the Central Nervous System During Neuroinflammation: A Novel Role for Endothelial-Derived Extracellular Vesicles

Debayon Paul et al. J Neuroinflammation. .
Free PMC article

Abstract

Background: The mechanism of leukocyte transendothelial migration (TEM) across the highly restrictive blood-brain barrier (BBB) remains enigmatic, with paracellular TEM thought to require leukocytes to somehow navigate the obstructive endothelial tight junctions (TJs). Transient interactions between TJ proteins on the respective leukocyte and endothelial surfaces have been proposed as one mechanism for TEM. Given the expanding role of extracellular vesicles (EVs) in intercellular communication, we investigated whether EVs derived from brain microvascular endothelial cells (BMEC) of the BBB may play a role in transferring a major TJ protein, claudin-5 (CLN-5), to leukocytes as a possible basis for such a mechanism during neuroinflammation.

Methods: High-resolution 3D confocal imaging was used to highlight CLN-5 immunoreactivity in the central nervous system (CNS) and on leukocytes of mice with the neuroinflammatory condition experimental autoimmune encephalomyelitis (EAE). Both Western blotting of circulating leukocytes from wild-type mice and fluorescence imaging of leukocyte-associated eGFP-CLN-5 in the blood and CNS of endothelial-targeted, Tie-2-eGFP-CLN-5 transgenic mice were used to confirm the presence of CLN-5 protein on these cells. EVs were isolated from TNF-α-stimulated BMEC cultures and blood plasma of Tie-2-eGFP-CLN-5 mice with EAE and evaluated for CLN-5 protein by Western blotting and fluorescence-activated cell sorting (FACS), respectively. Confocal imaging and FACS were used to detect binding of endothelial-derived EVs from these two sources to leukocytes in vitro. Serial electron microscopy (serial EM) and 3D contour-based surface reconstruction were employed to view EV-like structures at the leukocyte:BBB interface in situ in inflamed CNS microvessels.

Results: A subpopulation of leukocytes immunoreactive for CLN-5 on their surface was seen to infiltrate the CNS of mice with EAE and reside in close apposition to inflamed vessels. Confocal imaging of immunostained samples and Western blotting established the presence of CLN-5+ leukocytes in blood as well, implying these cells are present prior to TEM. Moreover, imaging of inflamed CNS vessels and the associated perivascular cell infiltrates from Tie-2-eGFP-CLN-5 mice with EAE revealed leukocytes bearing the eGFP label, further supporting the hypothesis CLN-5 is transferred from endothelial cells to circulating leukocytes in vivo. Western blotting of BMEC-derived EVs, corresponding in size to both exosomes and microvesicles, and FACS analysis of plasma-derived EVs from Tie-2-eGFP-CLN-5 mice with EAE validated expression of CLN-5 by EVs of endothelial origin. Confocal imaging and FACS further revealed both PKH-67-labeled EVs from cultured BMECs and eGFP-CLN-5+ EVs from plasma of Tie-2-eGFP-CLN-5 mice with EAE can bind to leukocytes. Lastly, serial EM and 3D contour-based surface reconstruction revealed a close association of EV-like structures between the marginating leukocytes and BMECs in situ during EAE.

Conclusions: During neuroinflammation, CLN-5+ leukocytes appear in the CNS, and both CLN-5+ leukocytes and CLN-5+ EVs are detected in the blood. As endothelial cells transfer CLN-5+ to leukocytes in vivo, and EVs released from BMEC bind to leukocytes in vitro, EVs may serve as the vehicles to transfer CLN-5 protein at sites of leukocyte:endothelial contact along the BBB. This action may be a prelude to facilitate TEM through the formation of temporary TJ protein bridges between these two cell types.

Keywords: Blood-brain barrier; Exosomes; Extracellular vesicles; Leukocytes; Microvesicles; Transendothelial migration.

Figures

Fig. 1
Fig. 1
CLN-5+ leukocytes are present in the CNS during early EAE. z-stack confocal images acquired from the same spinal cord cryosection of a WT mouse at D9 EAE are shown. a Immunostaining of TJ protein CLN-5 (green) and CD31 (red) to identify endothelial cells, showing CLN-5 is present on both leukocytes and at endothelial junctions. b DRAQ5 staining (blue) highlights the cellularity associated with CNS-infiltrating leukocytes, revealing CLN-5+ leukocytes (green) comprise a subset of invading cells. c Immunostaining of TJ protein CLN-5 only; arrows (yellow) indicate some CLN-5+ leukocytes are associated with areas of discontinuity of CLN-5 junctional staining. Leukocyte-associated CLN-5 immunoreactivity displays a punctate appearance
Fig. 2
Fig. 2
CLN-5+ leukocytes in circulation during early EAE. a Representative z-stack confocal images of PBLs isolated from WT mice at D8 EAE and immunostained with CLN-5 (green isosurface) under non-permeabilized or permeabilized (with Triton X-100) conditions. Leukocytes were obtained a day earlier than in Fig. 1, to ensure those CLN-5+ cells in the circulation had not yet all extravasated. b FACS analysis of leukocytes from D8 EAE mice immunostained with the same antibody shows CLN-5 staining under both non-permeabilized and permeabilized conditions. Leukocytes stained with an isotype antibody were used as control. c Western blot analysis of lysates from the same batch of leukocytes and the same antibody clone used in a and b showing a 23 kDa molecular weight band, consistent with the molecular weight of CLN-5
Fig. 3
Fig. 3
Appearance of eGFP-CLN-5+ leukocytes in Tie-2-eGFP-CLN-5 mice during EAE. a z-stack confocal images acquired from spinal cord cryosections from Tie-2-eGFP-CLN-5 mice showing distribution of eGFP-CLN-5 (green) in naïve venules and associated with an inflamed venule and perivascular leukocytes at D9 EAE. Inset shows DRAQ staining (blue) highlighting the extent of perivascular cellularity, representing leukocyte infiltrates and a minority fraction of eGFP-CLN-5+ leukocytes. The presence of eGFP-CLN-5+ leukocytes in the CNS is consistent with these cells having acquired eGFP-CLN-5 from endothelial sources. b PBLs were isolated from Tie-2-eGFP-CLN-5 mice (n = 5) at D8 post-EAE induction and then subjected to FACS analysis and percentage eGFP-CLN-5+ CD45+ recorded (right). Control PBLs from naïve, WT mice were used to set the gate (left)
Fig. 4
Fig. 4
WT/Tie-2-eGFP-CLN-5 chimeras highlight endothelial origin of leukocyte CLN-5. Bone marrow cells from WT, non-transgenic donor mice (CD45.1/CD45.2) were transplanted into lethally irradiated about 6-week-old Tie-2-eGFP-CLN-5 host mice (CD45.2) via retro-orbital injection. a At 10 weeks post-transplant, tail bleeds were performed to assess the efficacy of leukocyte substitution. FACS shows PBLs from non-irradiated, host eGFP-CLN-5 mice are all CD45.2+ (left), while those in chimeras are approx. 98% CD45.1+ CD45.2+ (right), indicating host PBLs were nearly entirely replaced. b Two days following confirmation of leukocyte substitution, EAE was induced in chimeras and age-matched non-irradiated, host eGFP-CLN-5 mice (at approx. 16 weeks of age; n = 4). At D8 EAE, PBLs were isolated from both groups of mice and percentages of eGFP-CLN-5+ CD45+ determined. Control PBLs from naïve, WT mice and PBLs labeled with isotype control antibodies were used to set the gates (data not shown). c z-stack confocal image from the spinal cord section of chimeric mouse at D9 EAE showing eGFP-CLN-5 (green) distribution along the intercellular boundaries of a CNS microvessel (arrows) and associated with an aggregate of perivascular leukocytes (arrowhead). Insert shows high-power field of aggregated eGFP-CLN-5+ leukocytes
Fig. 5
Fig. 5
CLN-5+ expression in endothelial-derived EVs. a, b Western blot analysis of CLN-5 in EVs from supernatants of TNF-α-treated BMEC cultures. Total EVs (a) and exosome- and microvesicle-size EVs separated by differential ultracentrifugation (b), showing a 23 kDa molecular weight band consistent with the molecular weight of CLN-5. c FACS analysis of total EVs isolated from blood plasma of Tie-2-eGFP-CLN-5 mice with EAE, showing eGFP-CLN-5+ EVs alone (left) or eGFP-CLN-5+ EVs co-stained with PKH-26 dye to label all (eGFP-CLN-5+ and eGFP-CLN-5) EVs (right), in the double-positive event gate. Approx. 0.4% of all blood-derived EVs are shown as eGFP-CLN-5+ PKH-26+. Nano-fluorescent-size standard beads (not shown) were used for setting the gates
Fig. 6
Fig. 6
EVs from endothelial cells bind to leukocytes in vitro. ac EVs collected from supernatants of TNF-α-treated BMEC cultures were incubated with isolated PBLs. a z-stack confocal images show PKH-67-labeled (green) exosome- and microvesicle-size EVs separated by differential ultracentrifugation can bind to PKH-26-labeled naïve PBLs (red), along with DRAQ5 staining of PBL nuclei (blue). Insets highlight co-localization of staining on a single PBL. bc Binding of total PKH-67-labeled EVs to naïve PBLs. FACS analysis revealed 4.5% of the PBLs were PKH-26+ PKH-67+ (double-positive), suggestive of binding. The percentage of PBLs binding to PKH-67+ EVs diminished by 93%, while the PKH-67 signal intensity per leukocyte (mean fluorescence intensity), a reflection of the relative EV binding/cell, decreased by 65% in the presence of a 500-fold excess of unlabeled EVs. d Total eGFP-CLN-5+ EVs, FACS-purified from blood plasma of Tie-2-eGFP-CLN-5 mice at D8 EAE, bind to naïve PBLs in vitro. In this case, PBLs are identified by CD45 staining (blue). Inset further highlights a punctate binding pattern of eGFP-CLN-5+ EVs to PBLs
Fig. 7
Fig. 7
EV-like structures in situ at sites proximal to leukocyte adhesion. Serial EM images from mouse spinal cord sections obtained at D13 EAE. a Single serial section showing cross section of an inflamed venule highlighting adherent leukocytes, some apparently undergoing TEM. Inset highlights EV-like membrane-bound structures (blue arrowhead) at the leukocyte-endothelial interface of an adherent leukocyte (blue box). b Representative 3D reconstruction of an adherent leukocyte shown in a, generated from 130 serial slices. The larger membranous structure indicated by * is on the order of 1 μm in diameter and may represent an apoptotic body loosely tethered to the leukocyte. c Contour surface reconstruction of the “traced” leukocyte (red), EV-like structures (green), and endothelium (turquoise) in all 130 serial slices, providing a 3D view of all three elements at the site of leukocyte docking (yellow asterisk). Inset provides an oblique view of the site of leukocyte attachment, showing EV-like structures and leukocyte process (yellow arrowheads) apparently below the surface of the endothelial plasma membrane. d, e 3D surface reconstruction of the leukocyte in panels ac, along with a representative serial EM slice, highlighting various patterns and sizes of EV-like structures, close to the site of leukocyte binding
Fig. 8
Fig. 8
Interactions of endothelial CLN-5+ EVs with leukocytes. EVs shed from endothelial cells could potentially transfer CLN-5 protein to leukocytes and foster TEM by several conceivable scenarios: [1] binding of shed CLN-5+ EVs to undefined sites on the leukocyte surface, [2] binding of nascent CLN-5+ EVs still associated with the endothelium to endogenous CLN-5 on the leukocyte surface, and [3] binding of shed CLN-5+ EVs to endogenous CLN-5 on the leukocyte surface, resulting in temporary cross-linking of leukocyte to the endothelium. Binding of CLN-5+ EVs to endogenous CLN-5 on the leukocyte surface could potentially amplify leukocyte:endothelial interactions by increasing avidity of CLN-5-binding partners. Not shown are possibilities EVs might inject endothelial-derived CLN-5 protein and/or mRNA into the leukocyte for surface expression. Concentrated release of EVs near the junctional region could act in a juxtacrine manner to guide leukocytes to this site for TEM

Similar articles

See all similar articles

Cited by 16 articles

See all "Cited by" articles

References

    1. Abbott NJ, Friedman A. Overview and introduction: the blood-brain barrier in health and disease. Epilepsia. 2012;53(Suppl 6):1–6. doi: 10.1111/j.1528-1167.2012.03696.x. - DOI - PMC - PubMed
    1. Bauer HC, Krizbai IA, Bauer H, Traweger A. “You shall not pass”-tight junctions of the blood brain barrier. Front Neurosci. 2014;8:392. doi: 10.3389/fnins.2014.00392. - DOI - PMC - PubMed
    1. Preston JE, Joan Abbott N, Begley DJ. Transcytosis of macromolecules at the blood-brain barrier. Adv Pharmacol. 2014;71:147–63. doi: 10.1016/bs.apha.2014.06.001. - DOI - PubMed
    1. Campos-Bedolla P, Walter FR, Veszelka S, Deli MA. Role of the blood-brain barrier in the nutrition of the central nervous system. Arch Med Res. 2014;45(8):610–38. doi: 10.1016/j.arcmed.2014.11.018. - DOI - PubMed
    1. Alvarez JI, Cayrol R, Prat A. Disruption of central nervous system barriers in multiple sclerosis. Biochim Biophys Acta. 2011;1812(2):252–64. doi: 10.1016/j.bbadis.2010.06.017. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback