The Role of Properdin in Cellular and Humoral Responses – in vitro and in vivo analyses
2012-02-03T11:20:34Z (GMT) by
Complement is part of the innate immune defence. It can be activated via three pathways, the classical pathway, the lectin pathway, and the alternative pathway. It comprises soluble factors and receptors. Properdin (Factor P) is a soluble component of the alternative complement pathway which acts as an important positive regulator of complement activation that stabilises the alternative pathway convertases (C3bBb) and C3bBb5b in the feedback loop of the alternative pathway, protecting them from rapid inactivation. The thesis is interested to see the role of properdin in cellular and humoral immune responses by in vitro and in vivo analyses. Firstly, properdin was examined for its global activity by characterisation of promoter activity of the human gene for properdin which involved using bioinformatics and molecular biology. The promoter activity was measured by dual-luciferase reporter system. The findings appeared to have activity in the 670bp properdin plasmid construct in U937 non-LPS transfection but the transfection upon LPS stimulation was not successful. Next, by using a properdin-deficient mouse line as a tool, it is interested to investigate the role of properdin in immunity by using properdin-deficient mice as model in pneumococcal vaccination studies, in vitro characterisation of dendritic cells and mycobacterium infectious studies in bone marrow-derived dendritic cells culture. In vaccination studies demonstrated that vaccination proved efficacious as both properdin-deficient and WT had increased in total IgM level and specific IgM level after the vaccination as measured by commercial ELISA for total IgM and specific ELISA for PPS2 IgM. In the absence of properdin, specific anti-polysaccharide antibodies of the IgM type are made. Vaccinated properdin-deficient mice do not differ from wild type in their immunoglobulin response to the pneumococcal polysaccharide vaccine. Meanwhile properdin-deficiency had a benefit in survival, independent of vaccination. For the in vitro characterisation of dendritic cells, dendritic cells are derived from bone marrow and spleen culture, and flow cytometry measured dendritic cells phenotype surface markers. Both bone marrow and spleen dendritic cells derived from properdin deficient mice are impaired to be activated and mature as dendritic cells compared to wild type mice. The study presently concludes that the presence of properdin is essential to allow dendritic cells to develop their activated phenotype and properdin is a relevant player in dendritic cell mediated immune response. Further investigation of function of generated BM-derived DC of WT and properdin-deficient towards Mycobacterium tuberculosis and Mycobacterium bovis BCG. In overall findings of mycobacterial viability, secretion of TNF-α and intracellular containment of mycobacterium in BMDC, it is concluded that properdin has no role of in the immune response of BM-derived DC towards Mycobacterium tuberculosis and Mycobacterium bovis BCG. In conclusion, having properdin is essential to help the complement system as part of defence mechanism against infection. Additionally, properdin could play a ‘double-edged’ role.