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Title: Characterisation of the expression and degradation of the pro-inflammatory cytokine interleukin 1
Author: Zahedi-Nejad, Maryam Sadat
ISNI:       0000 0004 2719 9451
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2012
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Inflammation plays a crucial role in protecting the host from infection and tissue injury. However, uncontrolled inflammation contributes to the pathogenesis of major auto-inflammatory diseases. Interleukin-1 (IL-1), a pleiotropic pro-inflammatory cytokine, is a pivotal mediator of many of these diseases. The best characterised IL-1 family members, IL-1α and IL-1β, are produced as precursor forms of 31 kDa in size. Both precursors are cleaved and secreted, activating transmembrane IL-1 receptors on IL-1-responsive cells. Many studies that focused on IL-1α have shown that the precursor and processed mature Ct peptide, as well as its N terminus (Nt) form, can elicit a signal. However, with IL-1β, only the processed mature Ct form is known to elicit an inflammatory response and no immunological activity has been attributed to Nt fragments of pro-IL-1β. Therefore, the first objective of this study was to produce recombinant human Nt-IL-1β fragments in bacterial and mammalian expression system to investigate their possible immunomodulatory functions. Recombinant His-tagged N-terminus fragments (10 and 14 kDa) of pro-IL-1β were cloned into the bacterial expression vector pET-22(+) and expressed in E. coli BL21(DE3) followed by purification using three consecutive columns (IMAC, SEC and AEC). Purification analysis of eluted proteins from columns indicated that the recombinant proteins were always co-purified with some other bacterial proteins. The Nt fragments of pro-IL-1β were cloned into the mammalian expression plasmid, pcDNA3.1(+). Expression of these proteins was monitored by transfection of two mammalian cell lines: Human Embryonic Kidney (HEK) 293 cells and monkey kidney cells (COS-7). No protein expression was observed with either construct. These limitations urged us to investigate the expression and degradation of endogenous IL-1 in vitro. Previous studies have shown that the transcription of cytokine genes in response to lipopolysaccharide (LPS) is usually rapid and begins to decline within a few hours after stimulation. The proteasome is the major cellular proteolytic apparatus and controls the turn-over of cellular proteins. We investigated the intracellular stability of IL-1α and IL-1β in LPS-stimulated mouse J774 macrophages and primary mouse bone marrow derived macrophages (BMDMs). Exposure of LPS-stimulated J774 and BMDMs to three different classes of proteasome inhibitors (peptide alhedyde (ALLN), peptide boronate (MG262) and non-peptide inhibitor (β-lactone)) prevented the degradation of intracellular IL-1α and IL-1β in a concentration and time dependent manner. Furthermore, the release of IL-1 into the culture media was not affected by any of these inhibitors in LPS-stimulated J774 cells. However, in LPS-stimulated BMDMs, β-lactone increased the release of both IL-1α and IL-1β and ALLN only increased IL-1α release into culture supernatant compared to control. MG262 had no effect on the release of either. These data suggest that the proteasome plays an important role in controlling the amount of IL-1α and IL-1β by restricting the intracellular levels of these cytokines in activated monocytes and macrophages. Therefore, this study provides evidence in support of the hypothesis that the proteasome is involved in the degradation of IL-1α and IL-1β and may offer a potential therapeutic target in inflammatory diseases.
Supervisor: Brough, David; Pelegrin, Pablo Sponsor: Self-financed
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Interleukin1 ; IL-1a and IL-1ß ; Proteasome ; Proteasome inhibitors Monocytes ; LPS