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

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-dn-H-Ras-transduced eosinophils were treated with IL-5, they exhibited a time- and dosage-dependent reduction in extracellular regulated kinase 1 and 2 activation and an inhibition of p90 Rsk1 phosphorylation and IL-5-mediated eosinophil survival in vitro. In contrast, Tat-dn-H-Ras did not inhibit CD11b upregulation or STAT5 tyrosine phosphorylation. These data demonstrate that Tat dominant-negative protein transduction can serve as an important and novel tool in studying primary myeloid cell signal transduction in primary leukocytes and can implicate the Ras-Raf-MEK-ERK pathway in IL-5-initiated eosinophil survival

Eosinophils in glioblastoma biology

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The development of this malignant glial lesion involves a multi-faceted process that results in a loss of genetic or epigenetic gene control, un-regulated cell growth, and immune tolerance. Of interest, atopic diseases are characterized by a lack of immune tolerance and are inversely associated with glioma risk. One cell type that is an established effector cell in the pathobiology of atopic disease is the eosinophil. In response to various stimuli, the eosinophil is able to produce cytotoxic granules, neuromediators, and pro-inflammatory cytokines as well as pro-fibrotic and angiogenic factors involved in pathogen clearance and tissue remodeling and repair. These various biological properties reveal that the eosinophil is a key immunoregulatory cell capable of influencing the activity of both innate and adaptive immune responses. Of central importance to this report is the observation that eosinophil migration to the brain occurs in response to traumatic brain injury and following certain immunotherapeutic treatments for GBM. Although eosinophils have been identified in various central nervous system pathologies, and are known to operate in wound/repair and tumorstatic models, the potential roles of eosinophils in GBM development and the tumor immunological response are only beginning to be recognized and are therefore the subject of the present review

Nucleotide receptor signalling and the generation of reactive oxygen species

Elevated levels of extracellular nucleotides are present at sites of inflammation, platelet degranulation and cellular damage or lysis. These extracellular nucleotides can lead to the activation of purinergic (nucleotide) receptors on various leukocytes, including monocytes, macrophages, eosinophils, and neutrophils. In turn, nucleotide receptor activation has been linked to increased cellular production and release of multiple inflammatory mediators, including superoxide anion, nitric oxide and other reactive oxygen species (ROS). In the present review, we will summarize the evidence that extracellular nucleotides can facilitate the generation of multiple ROS by leukocytes. In addition, we will discuss several potential mechanisms by which nucleotide-enhanced ROS production may occur. Delineation of these mechanisms is important for understanding the processes associated with nucleotide-induced antimicrobial activities, cell signalling, apoptosis, and pathology

The nucleotide receptor P2RX7 mediates ATP-induced CREB activation in human and murine monocytic cells

Nucleotide receptors serve as sensors of extracellular ATP and are important for immune function. The nucleotide receptor P2RX7 is a cell-surface, ligand-gated cation channel that has been implicated in many diseases, including arthritis, granuloma formation, sepsis, and tuberculosis. These disorders are often exacerbated by excessive mediator release from activated macrophages in the inflammatory microenvironment. Although P2RX7 activation can modulate monocyte/macrophage-induced inflammatory events, the relevant molecular mechanisms are poorly understood. Previous studies suggest that MAPK cascades and transcriptional control via CREB-linked pathways regulate the inflammatory capacity of monocytic cells. As P2RX7 promotes MAPK activation and inflammatory mediator production, we examined the involvement MAPK-induced CREB activation in P2RX7 action. Our data reveal that stimulation of multiple monocytic cell lines with P2RX7 agonists induces rapid CREB phosphorylation. In addition, we observed a lack of nucleotide-induced CREB phosphorylation in RAW 264.7 cells expressing nonfunctional P2RX7 and a gain of nucleotide-induced CREB phosphorylation in human embryonic kidney-293 cells that heterologously express human P2RX7. Furthermore, our results indicate that P2RX7 agonist-induced CREB phosphorylation is partly mediated via Ca2+ fluxes and the MEK/ERK system. Mechanistic analyses revealed that macrophage stimulation with a P2RX7 agonist induces CREB/CREB-binding protein complex formation, which is necessary for CREB transcriptional activation. Also, we demonstrate that P2RX7 activation induces a known CREB-dependent gene (c-fos) and that dominant-negative CREB constructs attenuate this response. These studies support the idea that P2RX7 stimulation can directly regulate protein expression that is not dependent on costimulation with other immune modulators such as LPS