Characterisation of blood myeloid dendritic cells in mannose binding lectin-sufficient and mannose binding lectin-deficient individuals

Melinda Dean (2009). Characterisation of blood myeloid dendritic cells in mannose binding lectin-sufficient and mannose binding lectin-deficient individuals PhD Thesis, School of Medicine, The University of Queensland.

       
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Author Melinda Dean
Thesis Title Characterisation of blood myeloid dendritic cells in mannose binding lectin-sufficient and mannose binding lectin-deficient individuals
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2009-03
Thesis type PhD Thesis
Supervisor Dr. Slavica Vuckovic
Prof. Robert L. Flower
Dr. Damon Eisen
Prof Derek N.J. Hart
Total pages 223
Total colour pages 32
Total black and white pages 191
Subjects 11 Medical and Health Sciences
Abstract/Summary Mannose binding lectin (MBL) belongs to the collectin family of soluble pattern recognition molecules that elicit diverse biologic activities. Via multiple carbohydrate-recognition domains (CRD), MBL binds to mannose and N-acetyl-glucosamine oligosaccharides present on the surface of bacteria, fungi and yeast. Following pathogen recognition, MBL activates the complement system via MBL associated serine proteases in a manner independent of antibody and C1 complex. Deficiency in function and level of MBL is found in 25% of otherwise apparently healthy individuals, representing the most prevalent innate immune deficiency. MBL deficiency is a risk factor for the development of infections in humans and mice. The role of MBL as a modulator of infection is complex. MBL deficiency may influence proinflammatory cytokine production, expression of leukocyte adhesion molecules, or vascular damage, during the course of infection. Given that dendritic cells (DC) are antigen presenting cells (APC) with potent capacity to respond to microbial stimulation, I hypothesized that MBL deficiency may be reflected in DC functions associated with microbial stimulation. Initially, I investigated the association of MBL with human immune cells and demonstrated that in both MBL-Sufficient (MBL-S) and MBL-Deficient (MBL-D) individuals, MBL was particularly associated with monocytes. RT-PCR analysis demonstrated MBL was not transcribed by monocytes or other immune cells investigated (T, B, and NK cells, CD11c+DC, immature monocyte derived DC [MoDC], LPS matured MoDC, and granulocytes), suggesting MBL association with the cell surface may be via an adapter or co-receptor. Magnetically separated monocytes but not MoDC bound exogenous purified human plasma MBL (hpMBL). Addition of hpMBL (5 -15 µg/mL) did not induce MoDC activation, and MBL added together with lipopolysaccharide (LPS) did not induce MoDC activation above the level induced by LPS only. In the second part of this study, I used the particulate MBL ligand zymosan (Zy) as a pathogenic stimulus in a whole blood model to gain a greater understanding of the consequences of MBL deficiency. I compared surface phenotype, inflammatory cytokine production and antigen presenting capacity of blood myeloid (M)DC of MBL-D and MBL-S individuals following stimulation with Zy and MBL opsonised Zy (MBL-Zy). Blood MDC in MBL-D individuals, unlike their counterpart in MBL-S individuals, displayed unique functional characteristics, including higher production of proinflammatory cytokines IL-6 and TNF-, but poor capacity for allo-T cell effector cell induction. It appeared that stimulation with MBL-Zy reduced elevated production of IL-6 but not TNF- by blood MDC in MBL-D individuals. In the third part, expression microarray analysis was utilised to provide broad information on the genes and potential signalling pathways involved in the MDC responses in MBL-D and MBL-S individuals following stimulation with Zy and MBL-Zy. MBL-S individuals demonstrated greater capacity to induce T cell and NK cell signalling pathways than MBL-D individuals. Further, MBL acted as a regulator of important inflammatory molecules, namely T-cell receptor zeta (CD247), IFN-γ and perforin 1. The data presented in this study provides novel information on blood MDC function in MBL-S and MBL-D individuals in response to pathogen stimulation, and provided insight into mechanisms involved in the increased frequency of infection observed in MBL-D individuals.
Keyword Mannose Binding Lectin
MBL
Dendritic Cells
Innate Immunity
Zymosan
IL-6
TNF-a
Microarray
Additional Notes Colour pages 57,58,59,60,61,62,74,88,95,100,108,112,116,118,120,131,136, 141,142,145,148,151,155,158,161,162,166-168,171,188,191

 
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Created: Wed, 04 Nov 2009, 12:07:43 EST by Miss Melinda Dean on behalf of Library - Information Access Service