CD18 Activation Epitopes Induced by Leukocyte Activation

Abstract

The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.

Modulation of CD11b/CD18 Adhesive Activity by Its Extracellular, Membrane-Proximal Regions

“…Although the underlying mechanism for inside-out signaling remains elusive, the conformational changes within the CD11b/ CD18 receptor that occur as a result of cell activation have been well documented (33,34,44,45). Relevant to this study, it was reported that PMA stimulation of peripheral leukocytes leads to the expression of a neo-epitope on CD11b/CD18, which can be probed by an activation-dependent mAb, MEM148 (34).…”

RhoA Is Involved in LFA-1 Extension Triggered by CXCL12 but Not in a Novel Outside-In LFA-1 Activation Facilitated by CXCL9

“…In addition, PMA induces phosphorylation of Thr 758 in the ␤ 2 chain of LFA-1, which can modulate LFA-1 interactions with the actin cytoskeleton (62,63). Previous studies tested the effect of PMA on LFA-1 activity after prolonged exposure periods (10 -30 min) and demonstrated that this stimulation induces LFA-1 conformational changes detected by various reporter mAbs (10,19,21,64). Importantly, although 1 min stimulation of T cells with PMA is sufficient to trigger rapid LFA-1 adhesiveness to ICAM-1, it does not induce conformational changes of LFA-1.…”

“…The anti-␤ 2 integrin extension epitope KIM127 was a gift from M. K. Robinson (UCB-Celltech, Slough, U.K.). The LFA-1 Idomain allosteric IDAS-specific inhibitor A308926 and its control compound A270412 were gifts from D. Staunton (21). TS2.4-Alexa568 was a gift from F. W. Luscinskas (Harvard Medical School, Boston, MA).…”

Requirement of open headpiece conformation for activation of leukocyte integrin αXβ2

“…7E4 could bind to bent α X β 2 , in agreement with the presence of the closed headpiece in the bent conformation of α X β 2 (4). TS1/18 also blocks β 2 integrin activation on intact cells (27,35). This mAb binds to the residues on the α1-and α7-helices in the βI domain (Fig.…”