Lysophosphatidylcholine induces the production of IL-1beta by human monocytes.

There is evidence for the presence of lysophosphatidylcholine (lysoPC) in oxidatively modified low density lipoprotein, human plasma and in atherosclerotic lesions. We studied the effect of lysoPC on the cytokine production by human monocytes. Among all the cytokines tested (IL-8, TNF alpha, MCP-1 and IL-1beta), we found that lysoPC most consistently stimulated human monocytes to produce IL-1beta in a dose and time dependent manner. Adherent monocytes were exposed to lysoPC in cell culture medium containing 0.5% bovine serum albumin. When exposed to lysoPC from 12.5 to 75 microM, the cellular content of IL-1beta increased 2-4 fold. Up to a concentration of 50 microM no cytotoxic effect could be seen. Over 50 microM there was evidence of toxicity. The level of IL-1beta reached its highest level at 24 h and then declined. At 48 h, the cell associated IL-1beta was low, but still the lysoPC stimulated cells produced 4.1 times more IL-1beta than controls. Also the IL-1beta mRNA was augmented by lysoPC in parallel with the IL-1beta protein levels. The stimulatory effect of lysoPC was dependent on its chain length. There was no effect on IL-1beta production when the acyl chain was shorter than 16. We also found that saturated lysoPC 18:0 stimulated IL-1beta production more than the monounsaturated lysoPC 18:1. Thus, the lysoPC in oxidatively modified LDL may stimulate the production of IL-1beta in macrophages, which may contribute to the inflammatory response in atherosclerotic tissue.

Identification and analysis of macrophage-derived foam cells from human atherosclerotic lesions by using a "mock" FL3 channel in flow cytometry.

Macrophages are one of the major cell types in atherosclerotic lesions. They are believed to play an important role in the pathogenesis and development of the lesion, but their functional state and phenotypic characteristics are not well understood. Using flow cytometry, we analyzed surface markers of macrophages extracted from tissue digests. However, conventional techniques were hampered by the abundance of cell debris and extracellular lipids, which co-localized with double-positive cells in all fluorescent plots. We therefore developed a method to overcome this problem by using a novel gating technique in multiparameter flow cytometry. This method utilized the third fluorescence channel (FL3) as a "mock" channel, since no antibody conjugated to an FL3-specific fluorochrome was added to the samples. Cells single-positive for macrophage-specific monoclonal antibodies (mAb) conjugated to phycoerythrin (PE) (FL2) were separated from non-specific fluorescent particles in the FL2 versus FL3 fluorescent plot and a region excluding debris could be set. This was then used as a gate to exclude debris also in the first fluorescence channel (FL1) vs. FL2 plot in which expression of a panel of activation markers identified by fluorescein isothiocyanate (FITC)-conjugated mAb was analyzed. Using this strategy, we were able to identify and analyze the phenotype of macrophages from human atherosclerotic lesions. We were also able to sort these cells and in this way obtained a preparation of macrophage-derived foam cells from the tissue with little contamination of debris.