Monoclonal anti-b-actin and nicotinamide were purchased from SigmaAldrich

Monoclonal anti-b-actin and nicotinamide were purchased from SigmaAldrich

n enhanced production by already producing cells. Discussion Inflammation has been identified as the underlying cause of atherosclerosis and a low-grade chronic inflammatory response in the arterial walls has been suggested to drive both the induction and progression of the disease. A better understanding of the mechanisms that cause inflammation in this setting is therefore important and may lay the grounds for new therapeutic approaches. Contrary to the pro-inflammatory cytokines, which play a pivotal role in inducing and maintaining inflammation, macrophage-produced apoE has been shown to play an equally important but protective role in atherosclerosis. Given its anti-inflammatory properties, we have here investigated how production and secretion of apoE is influenced by a number of cytokines, all of which may be present in atherosclerotic lesions. For the purpose, we used a newly developed ELISpot assay by which apoE secretion may be studied at the single cell level. Using this approach we could demonstrate a low but significant number of apoE-secreting cells in non-activated PBMC, which, similar to what has been observed earlier with macrophages, could be significantly enhanced in the presence of TGF-b. Secretion was essentially limited to monocytes and tests with isolated 12411425 monocyte subpopulations defined classical and intermediate monocytes as the main producers with few positive cells in the non-classical population. This is interesting not least in view of previous difficulties in distinguishing these subpopulations based on functional characteristics such as the secretion of cytokines. Non-classical monocytes have also been claimed to be the most mature form of monocytes and the most likely to leave the blood stream and entering into tissue. If correct, this suggests that the majority of extravasating monocytes have a limited capacity to produce and secrete apoE. As most previous knowledge about apoE secretion by immune cells is based on Halofuginone experiences from macrophage cultures, we also used the ELISpot assay to compare apoE secretion in monocytes and macrophages. As evident from this comparison, 15168218 many of the features seen with monocytes could also be reproduced in macrophages including the upregulatory effect of TGF-b and the corresponding downregulation with IFN-c and LPS. However, due to the activated state of these cells with an already existing secretion of apoE, the effects were not as distinct and readily detected as in monocytes. Therefore, we think that monocytes may not only provide a more accessible source of cells but that they are also more susceptible to different interventions. inhibited apoE production in monocytes had a similar regulatory effect on liver cells. To address this, we used the hepatoma cell line HepG2 as well as freshly isolated hepatocytes. As seen in figure S3A, HepG2 cells showed a spontaneous secretion of apoE that was not enhanced by TGF-b and was also unaffected by the addition of pro-inflammatory cytokines as well as LPS. The same was true for normal hepatocytes. However, when supernatants were analysed by ELISA a slight, but significant, inhibition could be seen after adding IFN-a and TNF-a. The ELISA in the figure was done on 20-hour supernatants, but similar results were observed with supernatants collected after 48 hours. ELISpot vs. ELISA From the experiments with macrophages and HepG 2 cells, ELISA could appear as the more sensitive of the two Inflammation and apoE Production in Monocytes Ap

Proton-pump inhibitor

Website: