Blockade of Focal Clustering and Active Conformation in β2-Integrin-Mediated Adhesion of Eosinophils to Intercellular Adhesion Molecule-1 Caused by Transduction of HIV TAT-Dominant Negative Ras

Abstract

We transduced dominant negative (dn) HIV TAT-Ras protein into mature human eosinophils to determine the signaling pathways and mechanism involved in integrin-mediated adhesion caused by cytokine, chemokine, and chemoattractant stimulation. Transduction of TAT-dnRas into nondividing eosinophils inhibited endogenous Ras activation and extracellular signal-regulated kinase (ERK) phosphorylation caused by IL-5, eotaxin-1, and fMLP. IL-5, eotaxin-1, or fMLP caused 1) change of Mac-1 to its active conformation and 2) focal clustering of Mac-1 on the eosinophil surface. TAT-dnRas or PD98059, a pharmacological mitogen-activated protein/ERK kinase inhibitor, blocked both focal surface clustering of Mac-1 and the change to active conformational structure of this integrin assessed by the mAb CBRM1/5, which binds the activation epitope. Eosinophil adhesion to the endothelial ligand ICAM-1 was correspondingly blocked by TAT-dnRas and PD98059. As a further control, we used PMA, which activates ERK phosphorylation by postmembrane receptor induction of protein kinase C, a mechanism which bypasses Ras. Neither TAT-dnRas nor PD98059 blocked eosinophil adhesion to ICAM-1, up-regulation of CBRM1/5, or focal surface clustering of Mac-1 caused by PMA. In contrast to β2-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for β1-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for β2-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.

Transcellular Secretion of Group V Phospholipase A2 from Epithelium Induces β2-Integrin-Mediated Adhesion and Synthesis of Leukotriene C4 in Eosinophils

“…The signaling and regulating mechanisms controlling movement of eosinophils play an important role in the inflammatory process. Focal clustering of CD11b plays a key role in firm adhesion (28). Our results demonstrate a distinct clustering of CD11b (Fig.…”

“…To determine the mechanism by which hVPLA 2 up-regulates ␤ 2 integrinmediated adhesion, eosinophils were stained with mAb directed against CD11b and focal clustering of CD11b expression was visualized by confocal microscopy (28). In these experiments, eosinophils were activated with buffer control, 10 Ϫ7 M hVPLA 2 , 10 Ϫ7 M hIIaPLA 2 , and 10 Ϫ7 M FMLP for 15 min, and cytoslides were prepared in duplicate (Cytospin 2; Shandon).…”

Blockade of Airway Inflammation and Hyperresponsiveness by HIV-TAT-Dominant Negative Ras

“…These findings suggest that blockade of Ras-mediated events, which causes ERK phosphorylation and up-regulates integrin adhesion (23), also attenuates Ag-induced airway inflammation and hyperresponsiveness in mice.…”

“…Generation of TAT dnRas protein was accomplished by a method we have described previously (23). Briefly, a cDNA fragment encoding dn H-Ras was amplified by PCR from the H-Ras cDNA (17N mutant) in pUSEamp (Upstate Biotechnology, Lake Placid, NY).…”

“…Ras signaling also mediates both IgE-activation of mast cells (20) and gene expression for IL-5 caused by occupation of the Fc⑀R1 receptor (21). Ras-induced extracellular signal-regulated kinase (ERK) 4 activation is also essential for integrin-mediated eosinophil adhesion caused by cytokine, chemokine, and chemoattractant stimulation (22,23). H-Ras has been reported to play a role in IL-5-mediated survival of eosinophils in vitro (24).…”