Mitogen-activated protein kinases in dendritic cell maturation and death
Dendritic cells (DC) sense infection in their microenvironment and undergo a dynamic process of changes in phagocytic capacity, morphology and migratory activity in order to induce optimum T cell immunity. The acquisition of these properties is termed "DC maturation". In this study, key selected aspects of this DC maturation process have been explored. One aspect of DC maturation is the signalling pathways that DC use to respond to micro-environmental signals. DC respond to conserved microbial structures in their micro-environment via pattern recognition receptors including Toll-like receptors (TLRs). Generally it is thought that this leads to activation of the mitogen-activated protein kinase (MAPK) pathways, p38, ERK, and JNK. JNK and p38 MAPK are also activated by reactive oxygen species, including hydrogen peroxide (H202), known to be present in the inflammatory DC milieu. Therefore the ability of H2O 2 to stimulate DC, or modulate their response to TLR ligands such as lipopolysaccharide (LPS) was examined. Activation of JNK, associated with inhibition of tyrosine phosphatases, is linked to induction of DC apoptosis. JNK inhibition partially protected the DC from the pro-apoptotic effects of H2O2. Furthermore, H2O2 and LPS synergise in inducing JNK activation, and DC apoptosis. These studies suggest that excessive DC maturation, which may induce pathogenic T cell activation may be limited by DC apoptosis. The second section of the thesis explores the mechanisms leading to MAPK activation upon TLR ligation. Biochemical studies demonstrated that mixed lineage kinase 3 (MLK3), a kinase not previously implicated in DC maturation, was phosphorylated upon TLR3 and 4, but not TLR2 ligation. Studies using a selective MLK inhibitor supported a role for the mixed lineage kinase (MLK) family of MAPK kinase kinase (MAP3K) proteins in regulation of DC signalling, phenotype and cytokine secretion, in response to TLR3 and 4, but not TLR2 ligation. Selective activation of MAP3K may therefore contribute to the heterogeneity and flexibility of DC/pathogen interaction. A further feature of the DC sensory role is antigen uptake, but the relationship between morphology and phagocytic capacity has not been defined. Therefore a simple and flexible assay to examine this relationship was developed. DC with long dendritic processes were less efficient at antigen internalisation than round DCs.