Loughlin, Alison Jane; (1993) Functional properties of microglia relevant to inflammation and demyelination and their regulation by cytokines. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Immunocytochemical and electron microscopic techniques have implicated microglia in the processes of inflammation and demyelination in the central nervous system (CNS). The activity of microglia and other cellular participants in these events is subject to modulation by the local action of cytokines. Interleukin-1 (IL-1), IL-2, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNFα) have all been identified in MS brain tissue. To facilitate the study of microglial functional properties in vitro an existing procedure for rapid isolation of microglia was further optomised to improve yields and shorten the isolation time. Isolated rat microglia produce IL-1 and IL-6 on stimulation in vitro with lipopolysaccharide (LPS) and this effect is down-regulated by IFN-γ. Using in vivo microdialysis techniques, IL-1 and IL-6, secreted in response to injury within the CNS, have been monitored and, on the basis of immunocytochemical analysis, microglia have been identified as the likely source of these cytokines. Functional assays were used to assess the regulatory influence (inhibitory / stimulatory) of individual or combinations of cytokines on indicators of microglial antigen presenting and phagocytic capacities in vitro, these being judged particularly relevant to the processes of inflammation and demyelination in vivo. Whilst microglial phagocytic capacity was sensitive to LPS and a broad range of individual cytokines (IFN-γ, TNFα, IL-1, IL-3, IL-4, GM-CSF, TGFβ1), of the cytokines tested only IFN-γ and TGFβ1 affected antigen presenting capacity (MHC class II expression). However the stimulatory effect of IFN-γ on microglial antigen presenting capacity was modulated by TNFα, IL-4, TGFβ1 and LPS. The combined effect of two cytokines appears to depend on the sequence in which the cell is exposed to them and their relative concentrations. Particularly striking was the shift between an additive stimulatory and a mutually antagonistic effect of IL-4 and IFN-γ on phagocytic capacity when the IL-4 concentration was increased. Differences in the responses of microglia and peritoneal macrophages to cytokine treatment indicate that different regulatory mechanisms operate in these two cell types. Use has been made of a foetal rat CNS aggregate culture system with the capacity to myelinate, demyelinate and remyelinate, to investigate the role of macrophages in and the effect of cytokines on these processes. This culture system simulates the in vivo situation in terms of cell-cell interactions. Enrichment of aggregate cultures with macrophages enhances the degree of myelination which occurs, while demyelination is induced in this system by treatment with IFN-γ, TNFα, IL-1α and LPS. Interestingly, whilst the extent of demyelination induced by cytokines is not affected by the presence of greater numbers of macrophages, antibody-mediated demyelination is more extensive in these than in normal aggregate cultures. Cytokines may be acting directly on myelin or oligodendrocytes as well as modulating effector cell behaviour. The effect of macrophage enrichment on antibody-mediated demyelination may be indicative of an Fc and/or complement receptor-mediated mechanism. These results suggest that different mechanisms operate in cytokine and antibody-mediated demyelination.

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