Johnson, MA; (2007) The role of mannose-binding lectin in health and disease. Doctoral thesis , UCL (University College London).

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Abstract

Mannose-binding lectin (MBL) is a collagenous lectin found in the serum of mammals and birds. In humans, it is believed to play an important role in innate immunity by binding to carbohydrates on the surface of microorganisms and then activating the complement system via MBL-associated serine proteases (MASPs). MBL may also interact directly with phagocytic cells to promote the opsonisation of bacteria and viruses. Serum concentrations of MBL are determined by three structural mutations in the exon 1 region of the MBL2 gene and by polymorphisms in the MBL2 promoter region. Approximately a third of the population have low levels of MBL and appear to have increased susceptibility to infection, especially in children and the immunocompromised. The work presented in this thesis has provided further information on the role of MBL in susceptibility to infection and also provided insights into the binding of MBL to microorganisms and how MBL can influence cell adhesion molecule expression and neutrophil opsonophagocytosis. Initially, MBL binding to defined lipopolysaccharide (LPS) mutants of Helicobacter pylori and LPS and lipid A mutants of Neisseria meningitidis was assessed by flow cytometry. Dramatic differences in binding were observed between different mutants indicating the importance of LPS structure in determining MBL binding. Analyses of the LPS structures involved revealed that MBL binding was not merely dictated by the presence or absence of known ligands for MBL in the LPS terminal regions. In addition, four Mycoplasma organisms as well as 10 different Proteus mirabilis clinical isolates were studied for MBL binding. There was no binding of the lectin to any Proteus organisms and A39 strain of Mycoplasma whereas Mycoplasma pneumoniae, hominis and orale did bind MBL avidly. All binding was carbohydrate-specific and calcium dependent. Next two different MBL ELISA procedures were compared and then assessed in parallel with immunonephelometry in order to establish the most accurate assay for measuring MBL concentrations. The AntibodyShop Oligomer MBL ELISA kit was chosen for subsequent studies as the levels determined by this assay correlated well with MBL2 haplotypes. Using this assay, MBL plasma concentrations and MBL2 genotypes were determined in an unselected UK child population (ALSPAC). Both coding and promoter MBL2 variants were shown to be significantly associated with MBL levels. In another study three cohorts of patients with meningococcal disease, severe sepsis and cystic fibrosis were investigated. 770 patients with meningococcal disease were genotyped for MBL variants and their haplotype related to susceptibility to the disease and the severity of the disease as assessed by mortality. MBL genotype had only a marginal effect on susceptibility to the infection. However, there was a significant relationship between MBL2 variants and survival from this disease (chi-square test). In an adult cohort of patients with sepsis, the relationship between MBL2 exon 1 and promoter -221 polymorphisms, plasma levels of the encoded protein, and the incidence and outcome of severe sepsis and septic shock were investigated. Individual plasma levels were variable and increased between days 1 and 7. The mortality rate was higher in those with MBL levels <1000 uA (47.2 vs. 22.2%, P = 0.05) (multiple logistic regression analysis). The exon 1 polymorphisms (A/O or O/O) were significantly more common in the patients with severe sepsis and septic shock than in normal healthy adults (54.6% vs. 39.7%, P = 0.001). There was no significant difference in MBL2 genotype or haplotype frequency between survivors and nonsurvivors. In the third cohort, 260 children were recruited from the paediatric CF clinic at the Royal Brompton Hospital during 2000 and early 2001 for the next study. In this group, low MBL levels were not associated with poor pulmonary function during childhood. There was no significant difference in predicted FEVi, FVC or the annual rate of decline between high, medium and low-expressing MBL haplotype groups. The effect of MBL on the expression of adhesion molecules by endothelial cells following bacterial stimulation was also studied. MBL used at physiological concentrations significantly reduced the expression of CD62E by endothelial cells after their incubation with both wild type and cpsD' Neisseria meningitidis organisms. MBL did not make any difference to the CD54 expression by HUVECs under similar conditions. The difference in meningococcal LPS structure as well as incubation time did not influence MBL effect on the expression of both adhesion molecules. The final results chapter describes experiments which show that MBL has an additive effect to other complement pathways during complement deposition on Staphylococcus aureus. This led to enhance neutrophil phagocytosis of the bacterium. The studies presented in this thesis provide further insight into the potential role and mechanisms of MBL in human disease.

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