Accuri C6 Flow Cytometer System
Originally published online as doi:10.1189/jlb.0309150 on July 1, 2009

Published online before print July 1, 2009
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(Journal of Leukocyte Biology. 2009;86:851-862.)
© 2009 Society for Leukocyte Biology

The extracellular domain of CD11d regulates its cell surface expression

William M. McKillop*,{dagger},{ddagger}, John W. Barrett*,{ddagger}, Stephen H. Pasternak{ddagger}, Bosco M. C. Chan{dagger} and Gregory A. Dekaban*,{dagger},{ddagger},1

* Biotherapeutics Research Laboratory and
{ddagger} Molecular Brain Research Group, Robarts Research Institute, London, Ontario, Canada; and
{dagger} Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada

1. Correspondence: Biotherapeutics Research Group, Robarts Research Institute, Room 2-12, 100 Perth Drive, London, Ontario, Canada N6A 5K8. E-mail: dekaban{at}robarts.ca

ABSTRACT

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 β2-integrin family. Current evidence indicates that CD11d/CD18 is regulated differently than other β2-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.

Key Words: Golgi retention • β2-integrin • CD18 • {alpha}D