title: Biology of interleukin-6 production by human natural killer cells
Creators: name: Al-Tae, Firas
Publisher: name: University of Liverpool Repository
Year: 2013

description: NK cells secrete a variety of immune-regulatory cytokines and chemokines such as interferon - γ (IFN-γ) and tumour necrosis factor- α (TNF-α). However, in some of infections where NK cells have an important role in the defence mechanism, IL-6 appears to play a significant anti-viral role. Considering the role of both IL-6 and NK cells in these infections raises the possibility that IL-6 secretion by NK cells could be a main defence mechanism against them. Morover, NK cell-mediated IL-6 secretion may provide a critical link between the innate and adaptive immune response of the host. Furthermore, changes in this pathway in various autoimmune diseases, for example rheumatoid arthritis (RA) may be relevant for the pathogenesis of these disorders. The results presented in this thesis demonstrated that peripheral blood NK cells from healthy individuals have the ability to secrete IL-6 after co-culture with target cells against which these cells are known to exhibit direct cytotoxicity (either K562 or HeLa cells). The described secretory response was rapid, with IL-6 being significantly higher as early as 1 hour in HeLa co-cultures compared to 6 hours in case of K562 co-cultures. These findings were further confirmed when NK cells were activated alone with high doses of IL-2 or with non specific chemical activators (PMA+ ionomycin). These experiments clearly showed that NK cells have the potential to secrete IL-6 following activation. To analyse whether IL-6 secretion in the co-culture experiments was the result of direct cell-cell interactions between NK cells and the target cells or was induced by the presence of soluble mediators, co-culture experiments were set up where the target cells were separated from NK cells using a 0.4 µm pore size inserts. Separating NK cells and target cells abolished increases in cytokine production proving that direct interaction between NK cells and target cells is necessary for triggering IL-6 production by NK cells, as the semi-permeable membrane of Transwell chambers allows for the free passage of soluble factors but prevents direct cell-cell contact. Investigating the activating pathway which triggers the secretion of IL-6 by NK cells was the next step. This aim was achieved by inducing NK cell activation with immobilized antibodies against NK cell activating receptors and assessing the effect of the engagement of these receptors on peripheral blood NK IL-6 gene expression and protein secretion by quantitative real time PCR and ELISA.The results demonstrated that NKG2D and NKp46 were the two main receptors involved in the IL-6 mRNA expression and secretion by NK cells. The final aim of this thesis was to evaluate the biological significance of this secretion through an in vitro experimental model. We hypothesized that IL-6 secreted by NK cells could contribute to the migration of other inflammatory and immune cells to the site of inflammation. This hypothesis was based on the observations of others that IL-6 could induce direct CD4+ T cell migration. To test this hypothesis, an in vitro transmigration assay using Transwell inserts with 8 and 3 µm pore size were used. Our results demonstrated that CD4+ T cell migration in response to NK cells was inhibited by about 30% in the presence of neutralizing antibody to IL-6. These results signify the relative biological importance of IL-6 induced secretion by NK cells. In conclusion NK cells can contribute to IL-6 secretion. Given that NK cells appear at inflammation sites at the earliest stages of the process, where the number of other cells with a potential to secrete IL-6 is low, it is possible that NK cell-mediated IL-6 secretion is essential in orchestrating and potentiating the later stages of the adaptive immune response.