AB0074 INTERRELATIONSHIP BETWEEN NICOTINIC ACETYLCHOLINE RECEPTOR AND CYTOKINE PRODUCTION NOTED FOLLOWING T-CELL ANTIGEN RECOGNITION AND ACTIVATION

Abstract

Background T cells express muscarinic and nicotinic acetylcholine receptors (mAChRs, nAChRs) that increase intracellular Ca2+ [1] on stimulation. The expression of these receptors on macrophages and their activation by vagal stimulation has recently been the focus for novel arthritis treatment [2]. Objectives Our aim in the present study was to assess the effect of various peptides, on cytokine production and nAChRs inhibition. Methods nAChR heterologous subunits were expressed in Xenopus oocytes and the inhibitory activity of various peptides at ACh-evoked currents were assessed. The effect of these peptides on T-cell antigen recognition and subsequent cytokine production was assessed using an antigen presentation assay (APA). Briefly, the 2B4.11 murine T cell hybridoma recognizing cytochrome c as the antigen was co-cultured with the antigen presenting B cell hybridoma line LK35.2 (I-Ek bearing) and pigeon cytochrome c in the absence or presence of peptide or several nAChRs antagonists, including mecamylamine (broad nAChR antagonist), waglerin-1 (α1β1eδ), α-bungarotoxin (α7), RgIA (α9α10), Vc1.1 (α9α10) and dihydro-β-erythroidine hydrobromide (α4β4 and α4β2). ELISA and real-time PCR were performed to measure cytokine protein levels and nAChRs T-cells mRNA express levels separately. Results At 10μM, peptide W32052 had modest 50-55 % inhibition of human (h) α3β2 and hα4β2 nAChR subtypes, and 35% inhibition at hα9α10. W32052 greatly inhibited chimeric rat α1β1δ-mouse e (85%) at 10μM. W32052 also inhibited IL-2, IL-6, TNF-α and GM-CSF production at 50μM in the APA. nAChRs antagonists, mecamylamine (100μM), RgIA (10nM), Vc1.1 (17.5μM) and dihydro-β-erythroidine hydrobromide (10μM) could decrease IL-2 production. However, waglerin-1 and α-bungarotoxin did not affect IL-2 production in the APA. Conclusion W32052, an antagonist of nAChR, inhibits cytokine production following antigen recognition suggesting that there is a close link between T-cell antigen activation, ion channel regulation mediated by AChR and cytokine production. Further experiments are in progress.