Hannah L Bazzard
Exploring peripheral blood mononuclear cells as the source of interleukin-6 in polymyalgia rheumatica
Bazzard, Hannah L
Polymyalgia rheumatica (PMR) is a chronic inflammatory condition which affects the elderly, causing aching and stiffness of the neck, shoulders and pelvis, as well as more systemic manifestations such as fever, malaise and fatigue. PMR shares symptoms with rheumatoid arthritis (RA) and both are associated with significantly elevated circulating concentrations of interleukin-6 (IL-6), which is thought to play a key role in the pathogenesis of these diseases. In RA, IL-6 is derived from synovial cells in the joints. In PMR, however, the source of IL-6 is unknown. PMR patients do not exhibit the same joint involvement as in RA but they do have elevated circulating IL-6 concentrations, thus, it was hypothesised that the source of IL-6 in PMR may be one of the circulating peripheral blood mononuclear cell (PBMC) types, which are all capable of producing IL-6.
Blood samples were taken from untreated PMR patients, RA patients with active disease and healthy controls (HC) of similar age and gender. To account for known circadian variations in circulating IL-6, samples were taken at a standard time. IL-6 was quantified in plasma and serum using cytometric bead array (CBA) and enzyme-linked immunosorbent assay. The biological activity of the IL-6 was tested for the first time in PMR using a B cell proliferation assay. Using immunostaining and flow cytometry, constitutive intracellular IL-6 was measured in CD3+, CD14+, CD19+, CD123+ and CD11c+ PBMCs. Intracellular IL-6 was also determined following PBMC stimulation in vitro, to determine potential differences in cell responsiveness. Concentrations of secreted IL-6 in the culture supernatants of resting and stimulated PBMC were determined by CBA in parallel cultures. Other cytokines were also quantified in order to examine PMR and RA pathologies more broadly. Finally, the results of the cytokine assays were compared with patient reported severity of fatigue and the four different components of fatigue (emotional, living, physical and cognitive).
Circulating IL-6 was significantly elevated above HC in both serum and plasma of PMR and RA patients, and this IL-6 was found to be biologically active. PBMC in all subjects constitutively produced low levels of intracellular IL-6 and very low concentrations of secreted IL-6 in parallel cultures. Overall, responses to in vitro stimulation were variable but no significant differences were observed between PMR, RA and HC samples. Secreted IL-6, in contrast, increased dramatically following stimulation of all cultures, suggesting intracellular staining may not reflect the secretory capability of these cells, but also confirming that there were no differences between PBMC responses of PMR, RA and HC groups. A significant correlation was observed between circulating IL-6 concentrations in PMR and RA patients, and physical fatigue, living fatigue and total fatigue. Broader cytokine analysis demonstrated that IL-6 alone was significantly elevated in PMR patients.
Taken together, circulatory IL-6 in active PMR is found to be elevated in the absence of other inflammatory cytokines. It is biologically active and correlates strongly with physical and living aspects of fatigue. Circulating PBMCs are not the source of this elevated IL-6 in PMR patients, suggesting that neutrophils, vascular endothelium or muscle tissue may instead be involved.
Bazzard, H. L. Exploring peripheral blood mononuclear cells as the source of interleukin-6 in polymyalgia rheumatica. (Thesis). University of the West of England
|Keywords||peripheral blood mononuclear cells, interleukin-6, inflammation, autoimmune, flow cytometry|
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