Expression and function of mixed lineage kinases in dendritic cells.

Dendritic cells (DCs) sense the presence of conserved microbial structures in their local microenvironment via specific pattern recognition receptors (PRRs). This leads to a programme of changes, which include migration and activation, and enables them to induce adaptive T cell immunity. Mitogen-activated protein kinases (MAPKs) are implicated in this response, but the pathways leading from PRR ligation to MAPK activation, and hence DC activation, are not fully understood. Recent studies in the nervous system have suggested that the mixed lineage kinase (MLK) family of MAPK kinase kinase proteins may be involved as an intermediary step between PRRs and MAPKs. Therefore, in this study, we have used a well-established DC model to explore the role of MLKs in these cells. Messenger RNA for MLKs 2, 3, 4 and DLK and protein for MLKs 2, 3 and DLK are found in DC. DC activation in response to model PRR ligands, such as LPS or poly (I:C), is accompanied by phosphorylation of MLK3. In contrast, another known PRR ligand, zymosan, induces little MLK3 phosphorylation. Inhibition of MLK activity using a pharmacological inhibitor, CEP11004, blocks p38 and Jun N-terminal kinase (JNK) MAPK activation in response to LPS and poly (I:C), but not zymosan. The inhibition is associated with a block in DC activation as measured by cell-surface marker expression and cytokine secretion. Thus, MLKs are expressed in DC, and are implicated in DC activation, and the involvement of MLKs appears to be selective, depending on the nature of the DC stimulus.

Mixed lineage kinases (MLKs): a role in dendritic cells, inflammation and immunity?

This review summarizes current knowledge about the mixed lineage kinases (MLKs) and explores their potential role in inflammation and immunity. MLKs were identified initially as signalling molecules in the nervous system. They were also shown to play a role in the cell cycle. Further studies documented three groups of MLKs, and showed that they may be activated via the c-Jun NH(2) terminal kinase (JNK) pathway, and by Rho GTPases. The biochemistry of the MLKs has been investigated in considerable detail. Homodimerization and heterodimerization can occur, and both autophosphorylation and autoinhibition are seen. The interaction between MLKs and JNK interacting protein (JIP) scaffolds, and the resultant effects on mitogen activated protein kinases, have been identified. Clearly, there is some redundancy within the MLK pathway(s), since mice which lack the MLK3 molecule are not abnormal. However, using a combination of biochemical analysis and pharmacological inhibitors, several recent studies in vitro have suggested that MLKs are not only expressed in cells of the immune system (as well as in the nervous system), but also may be implicated selectively in the signalling pathway that follows on toll-like receptor ligation in innate sentinel cells, such as the dendritic cell.

Dendritic cells in viral pathogenesis: protective or defective?

Dendritic cells (DC) are potent antigen-presenting cells that are critical in the initiation of immune responses to control and/or eliminate viral infections. Recent studies have investigated the effects of virus infection on the biology of DC. This review summarizes these changes, focusing on both the DC parameters affected and the viral factors involved. In addition, the central role of DC biology in the pathogenesis of several viral families, including herpesviruses, paramyxoviruses and retroviruses, is explored. The field of pathogen recognition by DC is addressed, focusing on its role in protecting the host from viral infection, as well as the ability of viruses to exploit such host receptor ligation and signalling to their replicative advantage. The hypothesis is proposed that virus and host have evolved a symbiotic relationship to ensure both viral transmission and host survival.

JNK activation limits dendritic cell maturation in response to reactive oxygen species by the induction of apoptosis.

Dendritic cells (DC) sense infection in their local microenvironment and respond appropriately in order to induce T cell immunity. This response is mediated in part via the mitogen-activated protein kinase (MAPK) pathways. Hydrogen peroxide is present frequently in the inflammatory DC milieu and is known to activate MAPK. Therefore this study examines the role of hydrogen peroxide, both alone and in combination with lipopolysaccharide (LPS), in the regulation of activation of two key MAPK, p38 and JNK, regulation of phenotype, and regulation of apoptosis in human monocyte-derived DC. At low concentrations, hydrogen peroxide activates p38, but does not alter DC phenotype. At higher concentrations, hydrogen peroxide activates both p38 and JNK. Activation of JNK, which is associated with inhibition of tyrosine phosphatases in DC, is linked to the induction of DC apoptosis. An upstream JNK inhibitor (CEP11004) and a competitive JNK inhibitor (SP600125) both partially protected the DC from the proapoptotic effects of hydrogen peroxide. Unexpectedly, hydrogen peroxide and LPS synergize in inducing JNK activation and DC apoptosis. JNK-mediated apoptosis may limit damaging immune responses against neoepitopes generated by modification of self-antigens by reactive oxygen species present at sites of inflammation.

The use of targeted microbeads for quantitative analysis of the phagocytic properties of human monocyte-derived dendritic cells.

The relationship between phagocytic capacity and morphology of dendritic cells (DCs) has not been investigated previously. Therefore, in order to approach this question, we have developed a novel assay, which is described here. The model of dendritic cells (DCs) used was based upon cytokine-induced differentiation of peripheral blood mononuclear cells, followed by culture on a fibronectin substratum. Under these conditions, standard current methods of quantifying phagocytosis are not applicable, as they rely upon flow cytometric analysis of fluid phase cells; and for adherent cells, quantitative efficiency of uptake is very difficult to measure. Furthermore, for both fluid phase and adherent cells, it is difficult to discriminate between internal and externally bound probe, and degradation of internalised probes can lead to artefacts. Therefore, in this study, these technical issues have been overcome by a simple and flexible assay. Phycoerythrin (PE)-conjugated antibodies are used to target microbeads to the DCs. Following an appropriate incubation period, secondary staining with fluorescein isothiocyanate (FITC)-conjugated antibody allows discrimination between internal and externally bound beads. Microscopic visualisation allows individual beads to be studied easily and thus phagocytosis quantified, whilst permitting parallel examination of morphological parameters. In particular, the relationship between bead uptake and the nature and distribution of the dendritic processes can be evaluated.

Herpes simplex virus type-1-induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type I IFN secretion.

Adaptive cellular immunity is required to clear HSV-1 infection in the periphery. Myeloid dendritic cells (DCs) are the first professional Ag-presenting cell to encounter the virus after primary and secondary infection and thus the consequences of their infection are important in understanding the pathogenesis of the disease and the response to the virus. Following HSV-1 infection, both uninfected and infected human DCs acquire a more mature phenotype. In this study, we demonstrate that type I IFN secreted from myeloid DC mediates bystander activation of the uninfected DCs. Furthermore, we confirm that this IFN primes DCs for elevated IL-12 p40 and p70 secretion. However, secretion of IFN is not responsible for the acquisition of a mature phenotype by HSV-1-infected DC. Rather, virus binding to a receptor on the cell surface induces DC maturation directly, through activation of the NF-kappaB and p38 MAPK pathways. The binding of HSV glycoprotein D is critical to the acquisition of a mature phenotype and type I IFN secretion. The data therefore demonstrate that DCs can respond to HSV exposure directly through recognition of viral envelope structures. In the context of natural HSV infection, the coupling of viral entry to the activation of DC signaling pathways is likely to be counterbalanced by viral disruption of DC maturation. However, the parallel release of type I IFN may result in paracrine activation so that the DCs are nonetheless able to mount an adaptive immune response.