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

Abstract

Chemokines presented on endothelial tissues instantaneously trigger LFA-1-mediated arrest on ICAM-1 via rapid inside-out and outside-in (ligand-driven) LFA-1 activation. The GTPase RhoA was previously implicated in CCL21-triggered LFA-1 affinity triggering in murine T lymphocytes and in LFA-1-dependent adhesion strengthening to ICAM-1 on Peyer’s patch high endothelial venules stabilized over periods of at least 10 s. In this study, we show that a specific RhoA 23/40 effector region is vital for the initial LFA-1-dependent adhesions of lymphocytes on high endothelial venules lasting 1–3 s. Blocking the RhoA 23/40 region in human T lymphocytes in vitro also impaired the subsecond CXCL12-triggered LFA-1-mediated T cell arrest on ICAM-1 by eliminating the rapid induction of an extended LFA-1 conformational state. However, the inflammatory chemokine CXCL9 triggered robust LFA-1-mediated T lymphocyte adhesion to ICAM-1 at subsecond contacts independently of the RhoA 23/40 region. CXCL9 did not induce conformational changes in the LFA-1 ectodomain, suggesting that particular chemokines can activate LFA-1 through outside-in post ligand binding stabilization changes. Like CXCL9, the potent diacylglycerol-dependent protein kinase C agonist PMA was found to trigger LFA-1 adhesiveness to ICAM-1 also without inducing integrin extension or an a priori clustering and independently of the RhoA 23/40 region. Our results collectively suggest that the 23/40 region of RhoA regulates chemokine-induced inside-out LFA-1 extension before ligand binding, but is not required for a variety of chemokine and non-chemokine signals that rapidly strengthen LFA-1-ICAM-1 bonds without an a priori induction of high-affinity extended LFA-1 conformations.

Cytohesin-1 controls the activation of RhoA and modulates integrin-dependent adhesion and migration of dendritic cells

“…By use of blocking peptides, Giagulli et al 15 had found that RhoA is required for leukocyte beta-2 integrin activation in endothelial arrest. These findings were recently confirmed and extended by Pasvolsky et al, 39 who showed that RhoA blocking peptides regulate intermediate affinity epitope expression of LFA-1 in T cells in flow. We now show that cytohesin-1 regulates the GTP loading of RhoA in leukocytes and we consistently report that both cytohesin-1 and RhoA mediate chemokine-induced activation epitope expression of CD18 in dendritic cells (Figure 2, Figure 6E, and data not shown).…”

Rho GTPases: Masters of T lymphocyte migration and activation

“…This supports the notion that in vivo, the main modification of ␣ L ␤ 2 leading to adhesion is due to a shift from a low to highaffinity conformation. Results in primary human T cells confirm this observation: a permeant peptide blocking the 23-40 region reduces the stable arrest on ICAM1 coating or HEV in vivo [44]. However, no role for the 92-119 region in ␤ 2 stable nor transient adhesion has been reported in primary human T cells.…”

The GTPase-activating protein ARAP3 regulates chemotaxis and adhesion-dependent processes in neutrophils

“…RhoA has been shown to regulate leukocyte integrin activation in several situations. [47][48][49][50] In thymocytes, RhoA was shown to be required for optimal integrin activation by Rap1, 51 indicating that RhoA can be part of Rap-dependent inside-out activation of integrins. Given that Rap regulates ARAP3 as a Rho GAP and that ARAP3-deficient neutrophils are characterized by heightened ␤2 integrin activity and hyperresponsiveness in adhesion-dependent processes, we speculate that Rap activates ARAP3 as a Rho GAP to fine-tune integrin activity in neutrophils.…”