Date of Award
MD (Medical Doctor)
Professor Richard Costello
Interaction between eosinophils and nerve cells is known to occur in conditions such as asthma. Within nerve cells this interaction increases intracellular signaling via mitogen activated protein (MAP) kinase phosphorylation and nuclear factor (NF) KB activation resulting in increased anti-apoptotic and cholinergic gene expression. Activated eosinophils release potentially toxic cationic granular proteins, including major basic protein (MBP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO) in an adhesion-dependent manner. MBP has been shown to induce dysfunction of nervecell M2 receptors inhibiting cholinergic negative feedback and, like whole eosinophils, protect nerve cells from apoptosis by up-regulating Bfl-1 expression in an (NF)KB dependent manner.
This study was designed to examine the effects of eosinophil granule proteins on cholinergic gene expression in IMR32 nerve cells and assess the interactive and intracellular mechanisms leading to these changes.
EPO caused up-regulation of both choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) gene expression. ChAT mRNA up-regulation occurred early and transiently with levels returning to baseline at 4 hours. VAChT up-regulation however was first observed at 4 hours and sustained up to 24 hours of co-incubation. ChAT and VAChT protein levels were also increased between 18 and 24 hours of co-incubation with VAChT levels remaining elevated up to 48 hours. MBP caused an up-regulationof VAChT at 24 hours but had no effect on ChAT expression. EDN failed to up-regulate either gene.
The mechanism of ECP interaction with the cell membrane was assessed using the cationic charge mimetic poly-L-arginine (PLA), charge counteraction with the anionic poly-L-glutamate (PLG) and cleavage of heparan sulphate moieties of glycosaminoglycans (GAG'S) with heparinase 1. PLA had no effect on either ChAT or
VAChT expression at any time point. However, both PLG and heparinase 1 reversed the up regulatory effect of EPO on ChAT and VAChT RNA and protein suggesting that ECP charge is essential but in itself insufficient to instigate intracellular cascasdes and gene up-regulation.
EPO was demonstrated to cause ERK activation by phosphorylation. Electrophoretic mobility shift assay (EMSA) showed activation of NF-KB in IMR-32 cells within 10 min of treatment with EPO. ERK inhibition led to complete reversal of all up regulatory effects of EPO on ChAT and VAChT RNA and protein at relevant time points.
Using confocal microscopy, cellular localization of fluorescently labeled EPO to IMR32 cells is seen very clearly as early as 1 hour. There is evidence of preferential interaction with certain areas of membrane likely representing GAG rich areas and evidence of internalization at later time points. PLG and heparinase 1 block this interaction suggesting that charge may play a synergistic role to ECP adhesion at GAG rich sites on the cell membrane.
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Akasheh N B. The effects of eosinophil cationic granule proteins on cholinergic gene expression in nerve cells [MD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2011.