La migración y sus tipos

Immature DC in the periphery are specialised for antigen uptake and consequently demonstrate high levels o f phagocytosis, macropinocytosis and receptor-mediated endocytosis (Sallusto and Lanzavecchia, 1995). Immature DC, adhered to FN to promote dendrite formation, exhibit a significant degree o f endocytosis (Figure 3.12). The endocytic tracer Texas Red-conjugated dextran first localised to the outer membrane o f the dendrite along the length o f the dendrite, although it was particularly concentrated at the membrane-rich tips. It was then subsequently taken up into the cell and became localised to vesicular structures, presumably early endosomes, within 10 minutes. The dextran was then transported down the length o f the dendrite over the next 50 minutes and accumulated in a perinuclear location close to the cell body, where late endosomes have been shown to be located (Alberts et al.,

1994), where it remained for the next 60 minutes. HLA-DR that was initially located on the cell membrane appeared to be internalised following incubation o f the DC with dextran but was initially located in separate vesicular structures within the dendrites . After 120 minutes HLA-DR and dextran became localised to the same compartments, presumably the MHC compartments. In vivo this would be expected to permit loading o f antigenic peptides on to MHC class II molecules to form peptide-MHC complexes that would subsequently be transported to the cell surface. In the absence o f stimulation, as for the DC used in this part o f the study, these complexes would be rapidly recycled. However, upon activation DC would migrate to the lymphoid tissues where their half-life would be dramatically increased, as would levels o f co­ stimulatory molecules, to permit efficient presentation to T cells (Watts and Amigorena, 2000).

Dendrite formation bv immature DC

Although the experiments carried out to investigate the endocytic capacity o f immature DC adhered to FN are preliminary, the results obtained lend support to the hypothesis that the morphology o f immature DC contributes to their extraordinary endocytic capacity. It is interesting to note that under these conditions immature DC are able to effectively endocytose antigen. The model presented in this study therefore not only serves as a useful in vitro model for in vivo dendrite formation by immature DC, but further provides a model for studying the endocytic capacity o f immature DC in vitro that could be translated to in vivo situations. Interestingly, the observation that immature DC adhered to FN use the whole length o f the dendrite to take up antigen, albeit more at the tips, suggests that DC dendrites may indeed function to increase the surface area over which endocytosis can occur. The endocytosed dextran always accumulated close to the cell body at the end o f the time course, which involved transport o f the dextran down the length o f the dendrite, suggesting that the whole length o f the dendrite is exploited to maximise uptake prior to transport to the MIIC compartments for processing and loading on to MHC class II molecules.

Chapter 4

Lack of dendrite formation by

mature DC

Lack o f dendrite formation bv mature DC

4.1 Introduction

In Chapter 3 it was demonstrated that immature PBMC-derived DC, adhered to FN, formed dendrites that were morphologically similar to those observed in vivo. These studies provided an important novel model o f DC dendrite formation and supported the notion that the dendrites o f immature DC have a role in maximising the efficiency with which antigen is endocytosed in the periphery.

As a result o f the findings o f the previous chapter it was therefore possible to go on to investigate the second aim o f this thesis, namely to study the mechanism o f formation of dendrites by LPS-matured DC on FN, an area o f research that has been largely neglected previously.

Upon exposure to the microbial product LPS, DC mature and consequently down- regulate their endocytic capacity and switch instead to an antigen presenting mode. Upon activation in vivo it is assumed that DC retract their dendrites as the first step in migration to the lymphoid tissues where they come in to contact with T cells. In the lymphoid tissues they once again form long, arborising dendrites and present antigen to T cells, in the form o f peptide-MHC complexes on their cell surface, subsequently stimulating the activation and proliferation o f antigen-specific T cells (Mellman et a l,

1998). In lymphoid tissues, DC morphology is believed to maximise contact between DC and T cells and allow the formation o f immuno-regulatory “clusters” within which cross-talk between T cells can take place (Mitchison et al, 1987).

In this study human PBMC-derived DC were cultured in vitro in the presence of LPS for 24 hours prior to seeding on FN. Under these conditions LPS-matured DC showed a markedly reduced ability to adhere to FN, lending support to the findings o f Jancic

et a l (1998) that demonstrated a reduction in the ability o f mature DC to bind to FN. Moreover, LPS-matured DC consistently failed to form dendrites on FN.

To confirm this further, the effect o f LPS dose and FN concentration were investigated and showed that the LPS effect on DC dendrite formation was dose- dependent but not linked to FN level. Despite this, functional studies using TRITC- conjugated dextran revealed that LPS-matured DC on FN retained a significant level o f endocytosis.

These studies highlight the plasticity o f DC and suggest that the switching off o f the ability to form dendrites on fibronectin may be a key feature o f DC maturation.

These studies had an important impact on the remainder o f the thesis, as the inability o f LPS-matured DC to form dendrites on FN meant that immature DC would have to be used to examine the role o f Rho GTPases in the control o f dendrite formation.

Lack o f dendrite formation bv mature DC

4.2 Results