Neutrophil transmigration under shear flow conditions in vitro is junctional adhesion molecule-C independent.
Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-alpha, IL-1beta, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN beta(2) integrins during transendothelial migration.
Sircar
Monica
M
Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Bradfield
Paul F
PF
Aurrand-Lions
Michel
M
Fish
Richard J
RJ
Alcaide
Pilar
P
Yang
Lin
L
Newton
Gail
G
Lamont
Deanna
D
Sehrawat
Seema
S
Mayadas
Tanya
T
Liang
Tony W
TW
Parkos
Charles A
CA
Imhof
Beat A
BA
Luscinskas
Francis W
FW
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D018821:CD18 Antigens / Q000378:metabolism; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D002465:Cell Movement; D007136:Immunoglobulins / Q000276:immunology / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000187:drug effects / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology; D016180:Lentivirus Infections / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000187:drug effects / Q000378:metabolism; D033081:Shear Strength; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D018821:CD18 Antigens; D015815:Cell Adhesion Molecules; D007136:Immunoglobulins; D053583:Interleukin-1beta; C486723:Jam3 protein, mouse; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1