A master thesis from Aalborg University
Interleukin-1α Induction in Human Macrophages
Author(s)
Term
4. term
Publication year
2014
Submitted on
2014-05-28
Pages
43 pages
Abstract
Inflammation is a complex biological process in response to foreign substances, and inflammatory cytokines are known to have an essential role in the initial part of the immune response. However, many inflammatory diseases are related to malfunctions in the inflammatory processes, thus investigations of cytokine responses are of great interest. This study aims to characterize the interleukin-1α (IL-1α) response in blood macrophages from healthy individuals by stimulation with lipopolysaccharides (LPS). Mononuclear cells were extracted from ten healthy donors and the kinetics of IL-1α was examined by stimulating the cells with LPS for a varying amount of time. RNA was extracted from the cells and cDNA synthesis was conducted prior to quantitative PCR, in order to investigate the transcription of IL-1α. The protein synthesis was examined by use of flow cytometry. Additionally, cDNA was used for DNA sequencing of the IL-1α gene to identify any eventual single nucleotide polymorphisms (SNPs). The transcription revealed great inter-individual variability, though in general a rapid increase in mRNA expression was observed within 2-4 hours following LPS stimulation. The response remained highly elevated throughout the 20 h investigated. No significant difference (P>0.05) was observed in the amount of mRNA when comparing the different times that the cells were stimulated in. This indicates that a regression does not occur within the 20 h. The protein synthesis of IL-1α revealed a slow but steady enhancement over time. This was indicated by a statistically significant increase in the fluorescence intensity of the macrophages between 0 and 4h, 4 and 8 h, and 8 and 20 h (P<0.000). DNA sequencing of the IL-1α gene demonstrated a known SNP in six of the ten subjects. No association was found between the presence of the SNP and the IL-1α response. This study revealed that an IL-1α response to LPS stimulation contains both individual variations and general characterizations, and it forms the basis of further study of IL-1α in health and disease.
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