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Title P2X7R-driven IL-1 responses in differentiated murine Dendritic Cells; comparison with Macrophages.
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Publication Date
Degree Level doctoral
University/Publisher University of Manchester
Abstract The P2X7R is a functionally distinct member of the P2X non-selective cation channels and has been implicated in the initiation of immune responses. One of the most extensively characterised immune responses of the receptor is to signal the rapid aggregation of the inflammasome complex and signal the release of IL-1β. These investigations have focused in providing direct comparisons of P2X7R-driven IL-1 responses between DC and mouse macrophages (peritoneal macrophages [PMΦ] and bone marrow derived macrophages [BM-MΦ]). Expression of the P2X7R has been identified in all three populations both at the transcriptional (P2X7A variant) and protein levels. Activation with lipopolysaccharide (LPS) (2h) induced a rapid dose dependent release of IL-6 but not of IL-1β in BM-DC. Rapid (2h) IL-1β release required both LPS priming and ATP activation. Both signals were also required for IL- 1β release in mouse ΒΜ-ΜΦ and PMΦ, however, at comparatively markedly lower levels. Furthermore, like with IL-1β, LPS did not induce IL-1α release in BM-DC. Interestingly, subsequent challenge with ATP evoked IL-1α release in BM-DC alone, with little or no detectable levels observed in activated BM-MΦ. This rapid IL-1β release (but not IL-6) was potently inhibited in both macrophages and DC with a P2X7R-specific inhibitor (A-740003) providing evidence that is predominantly a P2X7R-driven process. Treatment with A-740003 also potently inhibited IL-1α release from BM-DC suggesting that the ATP-P2X7R and caspase-1 activation might have a role in the release of the cytokine. Expression of gain-of-function P2X7K and loss-of- function P2X7J splice variants has been identified in both BM-DC and BM-MΦ, at the level of transcription. The possibility that a differential baseline or LPS-induced expression (at the transcriptional level) of P2X7J and P2X7K variants accounts for the diverse cytokine responses observed in BM-DC and BM-MΦ was also explored. However, the levels of expression for the various splice variants of interest (P2X7K and P2X7J) were found to be similar between the two cell types. The results of these investigations identify some subtle but intriguing differences in the mechanism of P2X7R activation and IL-1 release between DCs and macrophages. Purinergic signalling is increasing being implicated in the regulation of immune responses both in potentiating or suppressing inflammation. However, further work is required to decipher how the dynamic interplay between different purines can influence the immune activation of different cell types and indeed different cell subsets.
Subjects/Keywords P2X7R; Dendritic cells; Macrophages; IL-1beta; IL-1alpha; P2X7R splice variants
Contributors KIMBER, IAN I; Kimber, Ian; Dearman, Rebecca
Language en
Country of Publication uk
Format 333 page(s)
Record ID oai:escholar.manchester.ac.uk:uk-ac-man-scw-213941
Repository manchester
Date Retrieved
Date Indexed 2019-01-09

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…Figure 5.9: The impact of treatment with A-740003 on IL-1β release from LPS-primed and ATP challenged day 8 BM-DC and BM-MΦ. ....................................................... 230   Figure 5.10: The impact of treatment with A-740003 on IL-1α…

…and IL-6 release from LPS-primed and ATP challenged day 8 BM-DC and BM-MΦ. ................................... 232   Figure 5.11: The effect of apyrase treatment on IL-1 release from LPS-primed and ATP challenged day 8 BM-DC…

…induced changes in mRNA levels of P2X7R splice variant in BM-DC and BM-MΦ. ................................................................................................................. 238   Figure 5.14: The effect of apyrase treatment on IL-1β…

IL-1β release in day 8 BM-DC; comparisons with 8 BM-MΦ. .............................................. 242   Figure 5.16: Higher concentrations of LPS (24h) fail to induce IL-1β release in day 8 BM-DC; comparisons with day 8 BM-MΦ…

…244   Figure 5.17: The kinetics of LPS-induced IL-1β production and release in day 8 BMDC. ................................................................................................................................ 246   Figure 5.18…

…vein endothelial cells Id2 Inhibitor of DNA binding IFN- Interferon IGIF IFN-γ-inducing factor IL Interleukin IL-1RA Interleukin-1 receptor antagonist IL-1RacP IL-1 receptor accessory protein IL-1RI Type I Interleukin-1 receptor IL-1RII…

…inflammasome complex and signal the release of IL-1β. These investigations have focused in providing direct comparisons of P2X7R-driven IL-1 responses between DC and mouse macrophages (peritoneal macrophages [PMΦ] and bone marrow derived…

…rapid dose dependent release of IL-6 but not of IL-1β in BM-DC. Rapid (2h) IL-1β release required both LPS priming and ATP activation. Both signals were also required for IL1β release in mouse ΒΜ-ΜΦ and PMΦ, however, at comparatively markedly…

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