Transduction of a dominant-negative H-Ras into human eosinophils attenuates extracellular signal–regulated kinase activation and interleukin-5–mediated cell viability

Abstract

Inhibition of eosinophil apoptosis by exposure to interleukin-5 (IL-5) is associated with the development of tissue eosinophilia and may contribute to the inflammation characteristic of asthma. Analysis of the signaling events associated with this process has been hampered by the inability to efficiently manipulate eosinophils by the introduction of active or inhibitory effector molecules. Evidence is provided, using a dominantnegative N17 H-Ras protein (dn-H-Ras) and MEK inhibitor U0126, that activation of the Ras-Raf-MEK-ERK pathway plays a determining role in the prolongation of eosinophil survival by IL-5. For these studies, a small region of the human immunodeficiency virus Tat protein, a protein transduction domain known to enter mammalian cells efficiently, was fused to the N-terminus of dn-H-Ras. The Tat-dn-HRas protein generated from this construct transduced isolated human blood eosinophils at more than 95% efficiency. When Tat IntroductionAsthma is a complex syndrome of variable airflow obstruction, bronchial hyperresponsiveness, and airway inflammation characterized by the infiltration of eosinophils and increased levels of interleukin-5 (IL-5). IL-5 is the principal eosinophil-active cytokine, and it affects the differentiation, recruitment, viability, and cytotoxic effector functions of the eosinophil. Several signaling pathways are implicated in IL-5-mediated events, including the activation of the tyrosine kinases Lyn, Syk, and Jak2, 1-3 phosphorylation of signal transducers and activators of transcription (STAT) factors, 4-7 and the activation of the Ras-Raf-MEK-ERK pathway. 3,[8][9][10] Two lines of evidence suggest that IL-5 may activate extracellular signal-regulated kinase (ERK) 1 and ERK 2 through Ras-dependent pathways. First, in human peripheral blood eosinophils stimulated by IL-5, Ras is activated as measured by guanosine triphosphate loading. 9,11 In addition, experiments in the cytokine-dependent myeloid cell line BaF3 recapitulated the observations in cytokine-stimulated eosinophils when a constitutively active mutant of Ras transfected into the BaF3 cells promoted ERK activation and suppressed apoptosis. 12 These latter studies suggest that the Ras-Raf-MEK-ERK pathway may be important in IL-5-dependent suppression of apoptosis. Furthermore, a recent study has linked the survival of cerebellar neurons to the mitogen-activated protein (MAP) kinase pathway and reported that the ERK target protein, p90 ribosomal S6 kinase (p90 Rsk), could phosphorylate and inactivate the proapoptotic protein Bad and the cyclic adenosine monophosphateresponsive DNA-binding protein (CREB). 13 To establish the importance of a protein such as Ras in a signaling pathway, one approach is to render the protein or pathway inoperative. Multiple methods achieve the inhibition of cellular processes, including the use of pharmacologic agents to inhibit enzyme activity, the use of antisense oligonucleotides to reduce the mass of a given protein in a cell, or the introduction of dominantnegative (dn) mutant proteins...

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

“…7), suggesting that Ras not only is involved in Th2 cytokine production, but also in eosinophil migration induced by Th2 cytokines. Furthermore, Ras-induced ERK activation also is involved in IL-5-induced eosinophil survival (24). The relative contribution of each to the blockade of airway eosinophilic inflammation cannot be determined from this study.…”

“…The mechanism of these effects is unknown. Decreased eosinophil infiltration may result from one or more of the following: 1) decreased eosinophil adhesion to vascular endothelial cells (23), 2) decreased Th2 cytokine production, or 3) decreased eosinophil survival (24). We recently have demonstrated that the cytosolic group IV-PLA 2 and its upstream activators, ERK and Ras, regulate ␤ 2 -integrindependent adhesion of eosinophils in vitro (22,23).…”

“…Isolated leukocytes were resuspended in calcium-and magnesium-free HBSS (pH 6.8) containing 1% BSA. The pH of the sheath fluid of the flow cytometer was adjusted to 6.8 to quench FITC fluorescence on the outside of the cell (24), and at least 10,000 cells were analyzed on a FACScan (BD Biosciences, Mansfield, MA).…”

“…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). Recent investigations have reported that group IV cytosolic phospholipase A 2 (PLA 2 ), which is phosphorylated and activated by ERK (25), plays an essential role in Ag-induced eosinophil migration and airway hyperresponsiveness in immunestimulated mice (26) and guinea pigs (27).…”

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

“…However, the process by which cPLA 2 is activated to cause integrin-mediated adhesion remains unknown. HRas has been reported to cause attenuation of IL-5-mediated survival of eosinophils in vitro at 16 h (14). H-Ras also has been reported to enhance both ␤ 1 -and ␤ 2 -integrin-mediated adhesion in nondifferentiated cell systems (15,16).…”