The extracellular domain of CD11d regulates its cell surface expression.
A mAb targeting the CD11d subunit of the leukocyte integrin CD11d/CD18 decreases intraspinal inflammation and oxidative damage leading to improved neurological outcomes in rodent models of SCI. CD11d/CD18 is the fourth member of the beta2-integrin family. Current evidence indicates that CD11d/CD18 is regulated differently than other beta2-integrins, suggesting that CD11d(+) leukocytes play a distinct role in inflammation. Although the transcriptional control of CD11d expression has been evaluated, control of the intracellular distribution of CD11d has not been addressed. For this reason and as a result of the potential of CD11d as a therapeutic target for SCI and possibly other CNS injuries, we investigated the intracellular localization and surface expression of CD11d in cultured cells. CD11d and CD18 were fused at their C-termini with YFP and mRFP, respectively. Flow cytometry and confocal microscopy demonstrated that rCD11d-YFP is expressed on the cell surface of leukocyte cell lines expressing CD18. In contrast, in heterologous cell lines, CD11d-YFP is retained intracellularly in the TGN. Coexpression of CD11d-YFP and CD18-mRFP relieves this intracellular restriction and allows the CD11d/CD18 heterodimer to be surface-expressed. Based on domain-swapping experiments with CD25, the extracellular domain of CD11d is required and sufficient for the observed intracellular retention in heterologous cells. Furthermore, the transmembrane and C-terminus are also required for proper heterodimerization with CD18 and localization to the plasma membrane. These findings suggest that multiple CD11d domains play a role in controlling intracellular location and association with CD18.
McKillop
William M
WM
Biotherapeutics Research Laboratory, Robarts Research Institute, London, Ontario, Canada N6A 5K8.
Barrett
John W
JW
Pasternak
Stephen H
SH
Chan
Bosco M C
BM
Dekaban
Gregory A
GA
D000818:Animals; D018845:CD11 Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D018821:CD18 Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D019556:COS Cells; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D006801:Humans; D039001:Integrin alpha Chains / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D009187:Myelitis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D012377:Rodentia
D018845:CD11 Antigens; D018821:CD18 Antigens; C406812:ITGAD protein, human; D039001:Integrin alpha Chains