JAML mediates monocyte and CD8 T cell migration across the brain endothelium

Abstract

Leukocyte transmigration into the central nervous system promotes multiple sclerosis pathogenesis, yet ambiguity remains regarding the mechanisms controlling the migration of distinct immune cell subsets. Using in vitro, ex vivo and postmortem human materials, we identified a significant upregulation of junctional adhesion molecule-like expression at the blood-brain barrier, monocytes, and CD8 T cells of multiple sclerosis patients. We also detected junctional adhesion molecule-like(+) trans-migratory cups when monocytes/CD8 T cells adhered to the blood-brain barrier, however, their migratory capacity was significantly compromised when junctional adhesion molecule-like was blocked. These findings highlight a novel role for junctional adhesion molecule-like in leukocyte transmigration and its potential as a promising therapeutic target.

Figure 1

JAML expression on the BBB and immune cells during MS. qPCR (A) western blot (B) and immunocytofluorescent analysis (C) expression of JAML in primary cultures of resting and inflamed (TNF/IFN‐γ) human BBB‐ECs. Gene nomenclature for JAML is AMICA1. Data shown are representative of two to three independent experiments (n = 2–3 EC preparations), Scale bar: 10 μm (D) JAML expression was evaluated in MS tissues (both NAWM and lesions) and costained with the immune markers CD68 (macrophages/microglia), CD11c (dendritic cells), MHC‐II (antigen‐presenting cells) and CD3 (T cells), scale bars: 20 μm. (E and F) Representative flow cytometry dot plot showing the expression of JAML on human ex vivo CD14 monocytes and CD8 T cells in the peripheral blood of RRMS patients and controls. (G and H) MFI and percentage of JAML‐expressing CD14 monocytes and CD8 T cells in the peripheral blood of controls and RRMS patients and in the CSF of patients with active disease. (*P < 0.05, **P < 0.01, ***P < 0.001). JAML, junctional adhesion molecule‐like; BBB‐ECs, blood–brain barrier endothelial cells; MS, multiple sclerosis; NAWM, normal appearing white matter; CSF, cerebrospinal fluid; MFI, mean fluorescent intensity.

Figure 2

JAML support leukocyte migration across the BBB. (A and B) Expression of JAML (green) on CD14+ monocytes and CD8 T cells adhering to primary cultures of human BBB‐ECs. Costaining for p120 – ICAM‐1 (red) and nuclei (blue) are included. Y‐Z projections show JAML enhancement surrounding the leukocytes in transmigratory cups. Data shown are representative of four independent experiments. Scale bar = 10 μm. (C and D) Human ex vivo CD14 monocytes and activated CD8 T cells were allowed to migrate for 18 h across a monolayer of primary human BBB‐ECs pretreated with isotype control or anti‐JAML antibody. JAML blockade significantly restricted the migration of CD14 monocytes and activated CD8 T cells across BBB‐ECs. Data shown are representative of three independent experiments (n = 3 and 6 blood donors) performed in triplicate using two distinct BBB‐EC preparations (***P < 0.001). JAML, junctional adhesion molecule‐like; BBB‐ECs, blood–brain barrier endothelial cells.