Interleukin-1 potently contributes to 25-hydroxycholesterol-induced synergistic cytokine production in smooth muscle cell-monocyte interactions

Abstract

Objectives: Inflammation is essential for atherogenesis. Cholesterol, a cardiovascular risk factor, may activate inflammation in the vessel wall during this process. Cytokine-mediated interactions of human monocytes with vascular smooth muscle cells (SMCs) may perpetuate this process. Methods: We investigated the capacity of the cholesterol metabolite 25-hydroxycholesterol to induce inflammatory mediators in cocultures of freshly isolated monocytes with SMCs. We determined the role of interleukin-(IL)-1 in this interaction using qPCR, bioassays, ELISA and western blot. Cocultures with SMC to monocyte ratios from 1:4 to 1:20 were tested. Results: In separate SMC and monocyte cultures (monocultures) 25-hydroxycholesterol only poorly activated IL-1, IL-6 and MCP-1 production, whereas LPS stimulated much higher cytokine levels than unstimulated cultures. In contrast, cocultures of SMCs and monocytes stimulated with 25-hydroxycholesterol produced hundredfold higher cytokine levels than the corresponding monocultures. Blocking experiments with IL-1-receptor antagonist showed that IL-1 decisively contributed to the 25-hydroxycholesterol-induced synergistic IL-6 and MCP-1 production. The presence of intracellular IL-1β precursor, released mature IL-1β, and caspase-1 p10 indicated that the inflammasome was involved in this process. Determination of IL-1-mRNA in Transwell experiments indicated that the monocytes are the major source of IL-1, which subsequently activates the SMCs, the primary source of IL-6 in the coculture. Conclusion: Taken together, these interactions between local vessel wall cells and invading monocytes may multiply cholesterol-triggered inflammation in the vessel wall, and IL-1 may play a key role in this process. The data also indicate that lower cholesterol levels than expected from monocultures may suffice to initiate inflammation in the tissue.