Protein array technology to investigate cytokine production by monocytes from patients with advanced alcoholic cirrhosis: Anex vivopilot study

Aim: In patients with advanced cirrhosis, little is known about the ability of peripheral blood monocytes to spontaneously produce signaling proteins such as cytokines. The aim of this ex vivo study was to evaluate cytokine production under baseline conditions and after stimulation by lipopolysaccharide (LPS), a toll-like receptor (TLR) agonist. Methods: Peripheral blood monocytes were isolated from patients with advanced alcoholic cirrhosis (without ongoing bacterial infections) and normal subjects. Cells were left unstimulated or were stimulated with LPS. The abundance of 24 cytokines was measured using a filter-based, arrayed sandwich enzyme-linked immunosorbent assay (ELISA) in the supernatant of cultured monocytes. Results: Cirrhotic monocytes spontaneously produced six proteins (TNF-α, IL-6, IL-8, MCP-1, RANTES and Gro), whereas normal monocytes produced only small amounts of IL-8 and RANTES. Analyses with the online gene set analysis toolkit WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt) found enrichment for the six proteins in the human gene ontology subcategory (http://www.geneontology.org), Kyoto Encyclopedia of Genes and Genome pathways (http://www.genome.ad.jp/kegg/) and BioCarta pathways (http://www.biocarta.com/genes/index.asp) consistent with a proinflammatory phenotype of cirrhotic monocytes resulting from activated TLR signaling. Interestingly, LPS-elicited TLR engagement further increased the production of the six proteins and did not induce the secretion of any others, in particular the anti-inflammatory cytokine IL-10. LPS-stimulated normal monocytes produced TNF-α, IL-6, IL-8, MCP-1, RANTES, Gro and IL-10. Conclusion: In patients with advanced cirrhosis, peripheral blood monocytes spontaneously produce proinflammatory cytokines, presumably in response to unrestricted TLR signaling.