Jennifer L. Bankers-Fulbright

Interleukin-1 signal transduction

Interleukin-1 (IL-1) is primarily an inflammatory cytokine, although it is capable of mediating a wide variety of effects on many different cell types. Nearly every known signal transduction pathway has been reported to be activated in response to IL-1. However, the significance of many of these signaling events is unclear, due to the use of different and sometimes unique cell lines in studying IL-1-initiated signal transduction. Complicating matters further is the lack of association in many studies between identified IL-1-induced signals and subsequent biological responses. In this article, we review what is known about IL-1 receptor signaling and, whenever possible, correlate signaling events to biological responses.

Regulation of human eosinophil NADPH oxidase activity: A central role for PKCδ

Eosinophils play a primary role in the pathophysiology of asthma. In the lung, the activation state of the infiltrating eosinophils determines the extent of tissue damage. Interleukin‐5 (IL‐5) and leukotriene B4 (LTB4) are important signaling molecules involved in eosinophil recruitment and activation. However, the physiological processes that regulate these activation events are largely unknown. In this study we have examined the mechanisms of human eosinophil NADPH oxidase regulation by IL‐5, LTB4, and phorbol ester (PMA). These stimuli activate a Zn2+‐sensitive plasma membrane proton channel, and treatment of eosinophils with Zn2+ blocks superoxide production. We have demonstrated that eosinophil intracellular pH is not altered by IL‐5 activation of NADPH oxidase. Additionally, PKCδ inhibitors block PMA, IL‐5 and LTB4 mediated superoxide formation. Interestingly, the PKCδ‐selective inhibitor, rottlerin, does not block proton channel activation by PMA indicating that the oxidase and the proton conductance are regulated at distinct phosphorylation sites. IL‐5 and LTB4, but not PMA, stimulated superoxide production is also blocked by inhibitors of PI 3‐kinase indicating that activation of this enzyme is an upstream event common to both receptor signaling pathways. Our results indicate that the G‐protein‐coupled LTB4 receptor and the IL‐5 cytokine receptor converge on a common signaling pathway involving PI 3‐kinase and PKCδ to regulate NADPH oxidase activity in human eosinophils.

Regulation of eosinophil membrane depolarization during NADPH oxidase activation

Protein kinase C (PKC) activation in human eosinophils increases NADPH oxidase activity, which is associated with plasma membrane depolarization. In this study, membrane potential measurements of eosinophils stimulated with phorbol ester (phorbol 12-myristate 13-acetate; PMA) were made using a cell-permeable oxonol membrane potential indicator, diBAC4(3). Within 10 minutes after PMA stimulation, eosinophils depolarized from– 32.9±5.7 mV to +17.3±1.8 mV. The time courses of depolarization and proton channel activation were virtually identical. Blocking the proton conductance with 250 μM ZnCl2 (+43.0±4.2 mV) or increasing the proton channel activation threshold by reducing the extracellular pH to 6.5 (+44.4±1.4 mV) increased depolarization compared with PMA alone. Additionally, the protein kinase C (PKC) δ-selective blocker, rottlerin, inhibited PMA-stimulated depolarization, indicating that PKCδ was involved in regulating depolarization associated with eosinophil NADPH oxidase activity. Thus, the membrane depolarization that is associated with NADPH oxidase activation in eosinophils is sufficient to produce marked proton channel activation under physiological conditions.

Common Variable Immunodeficiency: Test Indications and Interpretations

Common variable immunodeficiency (CVID) is a primary immunodeficiency disorder that can present with multiple phenotypes, all of which are characterized by hypogammaglobulinemia, in a person at any age. A specific genetic defect that accounts for all CVID phenotypes has not been identified, and it is likely that several distinct genetic disorders with similar clinical presentations are responsible for the observed variation. In this review, we summarize the known genetic mutations that give rise to hypogammaglobulinemia and how these gene products affect normal or abnormal B-cell development and function, with particular emphasis on CVID. Additionally, we describe specific phenotypic and genetic laboratory tests that can be used to diagnose CVID and provide guidelines for test interpretation and subsequent therapeutic intervention.

Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils

The effects of platelet-activating factor (PAF) and IL-5 on intracellular pH were investigated in human eosinophils. Purified peripheral blood eosinophils were loaded with the ratiometric fluorescent pH indicator BCECF-AM ester. Stimulation of eosinophils with PAF produced time-dependent alkalinization of the cytoplasm from an initial pH of 7.1±0.04 to 7.5±0.05. A similar alkalinization response was produced by the calcium ionophore, ionomycin and by the calcium ATPase inhibitor, thapsigargin. These compounds as well as PAF produce significant increases in cytoplasmic calcium ([Ca2+]i). In contrast, IL-5 and the protein kinase C (PKC) activator phorbol myristate acetate (PMA) did not produce cytoplasmic alkalinization and had no effect on [Ca2+]i in eosinophils. PAF-stimulated alkalinization was not inhibited under conditions that blocked plasma membrane Na+-H+ exchange, proton channel or plasma membrane H+-ATPase activities. Measurements of intragranule pH with a cell permeant pH indicator (LysoSensor Yellow/Blue DND-160), which partitions into intracellular acidic compartments, revealed that PAF-stimulated cytosolic alkalinization correlated with intragranule acidification. These results suggest that the increase in [Ca2+]i after PAF stimulation activates a H+-ATPase present in the granule membranes, leading to enhanced granule acidification and cytoplasmic alkalinization. We propose that granule acidification is an important step in solubilization of major basic protein crystals, which are stored within the granule core, in preparation for degranulation and release of these proteins.

Novel Glucocorticomimetic Agents Active on Eosinophils: Potential Uses in the Therapy of Asthma

Publisher Summary It is believed that a KATP channel is present on the eosinophil membrane. By analogy with other KATP channels, a SUR and a Kir comprise the channel. The inhibition of IL-5-mediated eosinophil survival by lidocaine and by the SUR blockers is not overcome by increased IL-5 concentrations (up to 10,000 pg/ml), unlike the inhibition by glucocorticoids. It would seem most likely that the mechanism of action of lidocaine involves an inwardly rectifying potassium channel (Kir). The ability of KATP blockers to inhibit, and of openers to potentiate, IL-5-mediated survival is in keeping with the presence of an eosinophil SUR. A SUR family has recently been identified, and both SUR1 and SUR2 associate with and regulate Kir6.2. The two identified members, SUR1 and SUR2, are structurally related, but functionally distinct in that the SUR2-Kir6.2 combination is less sensitive to glyburide than the SUR1-Kir6.2 complex. Based on the concentrations of glyburide required to inhibit eosinophil survival in the experiments (c. 10-4 M), it is speculated that glyburide is not acting through the classic high-affinity SUR1, but rather functions through an analogous family member with a lower affinity for glyburide, possibly SUR2.

Novel Glucocorticomimetic Agents Active on Eosinophils

Publisher Summary It is believed that a KATP channel is present on the eosinophil membrane. By analogy with other KATP channels, a SUR and a Kir comprise the channel. The inhibition of IL-5-mediated eosinophil survival by lidocaine and by the SUR blockers is not overcome by increased IL-5 concentrations (up to 10,000 pg/ml), unlike the inhibition by glucocorticoids. It would seem most likely that the mechanism of action of lidocaine involves an inwardly rectifying potassium channel (Kir). The ability of KATP blockers to inhibit, and of openers to potentiate, IL-5-mediated survival is in keeping with the presence of an eosinophil SUR. A SUR family has recently been identified, and both SUR1 and SUR2 associate with and regulate Kir6.2. The two identified members, SUR1 and SUR2, are structurally related, but functionally distinct in that the SUR2-Kir6.2 combination is less sensitive to glyburide than the SUR1-Kir6.2 complex. Based on the concentrations of glyburide required to inhibit eosinophil survival in the experiments (c. 10-4 M), it is speculated that glyburide is not acting through the classic high-affinity SUR1, but rather functions through an analogous family member with a lower affinity for glyburide, possibly SUR2.

Chapter 19 – Novel Glucocorticomimetic Agents Active on Eosinophils: Potential Uses in the Therapy of Asthma

Publisher Summary It is believed that a KATP channel is present on the eosinophil membrane. By analogy with other KATP channels, a SUR and a Kir comprise the channel. The inhibition of IL-5-mediated eosinophil survival by lidocaine and by the SUR blockers is not overcome by increased IL-5 concentrations (up to 10,000 pg/ml), unlike the inhibition by glucocorticoids. It would seem most likely that the mechanism of action of lidocaine involves an inwardly rectifying potassium channel (Kir). The ability of KATP blockers to inhibit, and of openers to potentiate, IL-5-mediated survival is in keeping with the presence of an eosinophil SUR. A SUR family has recently been identified, and both SUR1 and SUR2 associate with and regulate Kir6.2. The two identified members, SUR1 and SUR2, are structurally related, but functionally distinct in that the SUR2-Kir6.2 combination is less sensitive to glyburide than the SUR1-Kir6.2 complex. Based on the concentrations of glyburide required to inhibit eosinophil survival in the experiments (c. 10-4 M), it is speculated that glyburide is not acting through the classic high-affinity SUR1, but rather functions through an analogous family member with a lower affinity for glyburide, possibly SUR2.