Melanoma cell adhesion molecule identifies encephalitogenic T lymphocytes and promotes their recruitment to the central nervous system

Abstract

In multiple sclerosis, encephalitogenic CD4(+) lymphocytes require adhesion molecules to accumulate into central nervous system inflammatory lesions. Using proteomic techniques, we identified expression of melanoma cell adhesion molecule (MCAM) on a subset of human effector memory CD4(+) lymphocytes and on human blood-brain barrier endothelium. Herein, we demonstrate that MCAM is a stable surface marker that refines the identification of interleukin 17(+), interleukin 22(+), RAR-related orphan receptor γ and interleukin 23 receptor(+) cells within the CD161(+)CCR6(+) subset of memory CD4(+) lymphocytes. We also show that MCAM(+) lymphocytes express significantly more granulocyte/macrophage colony stimulating factor and granzyme B than MCAM(-) lymphocytes. Furthermore, the proportion of MCAM(+) CD4(+) lymphocytes is significantly increased in the blood and in the central nervous system of patients with multiple sclerosis and experimental autoimmune encephalomyelitis animals compared with healthy controls or other neurological diseases, and MCAM expression is upregulated at the blood-brain barrier within inflammatory lesions. Moreover, blockade of MCAM or depletion of MCAM(+) CD4(+) T lymphocytes both restrict the migration of T(H)17 lymphocytes across blood-brain barrier endothelial cells and decrease the severity of experimental autoimmune encephalomyelitis. Our findings indicate that MCAM could serve as a potential biomarker for multiple sclerosis and represents a valuable target for the treatment of neuroinflammatory conditions.

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

“…Human BBB‐ECs were cultured to confluency and then treated for 18 h with TNF and IFN‐gamma, cells were trypsinized and RNA was isolated as described before 7, 8. RNA was reverse‐transcribed using Life Technologies(Grand Island, NY) high‐capacity cDNA reverse transcription kit following manufacturer's recommendations.…”

“…MS patients were classified as previously described 8. peripheral blood mononuclear cells (PBMCs) were isolated, stained, and analyzed as previously reported 7.…”

“…Primary cultures of BBB‐ECs were grown to confluency, inflamed, and stained as previously described 8. In brief, BBB‐ECs ( n = 3) were stained with anti‐JAML antibody (R&D systems, 1/50), followed by donkey anti goat‐Alexa 488 (Jackson ImmunoResearch‐West grove, PA).…”

“…In brief, BBB‐ECs ( n = 3) were stained with anti‐JAML antibody (R&D systems, 1/50), followed by donkey anti goat‐Alexa 488 (Jackson ImmunoResearch‐West grove, PA). Immunohistofluorescent stainings in postmortem brain sections from MS patients ( n = 5) were performed according to institutional guidelines (CRCHUM, SL05.022, SL05.023, and BH07.001) 8. Postmortem frozen MS brain blocks ( n = 24) were cryosectioned, fixed, and immunostained with goat anti‐JAML (R&D systems, 1/50) and with mouse anti‐CD68 (DAKO, 1/100), mouse anti‐CD11c (BD Biosciences, 1/200), rabbit anti‐CD3 (DAKO, 1/200) and mouse anti‐MHC‐II (DAKO, 1/100) followed by corresponding secondary antibodies (Jackson ImmunoResearch ‐ West Grove, PA).…”

“…Cells were then washed, fixed, and immunostained for JAML. intercellular adhesion molecule‐1 (ICAM‐1) (mouse anti‐ICAM1, Biolegend, San Diego ‐ CA) and p120 (mouse anti‐p120, BD Biosciences 1/100) as described before 8. To enable investigation of leukocyte migration across the BBB, we used a transwell model in which BBB‐ECs were grown on the upper chamber for 72 h 6, 7, 8.…”