Human endothelial cells express NOD2/CARD15 and increase IL-6 secretion in response to muramyl dipeptide

Mutations in the human NOD2/CARD15 gene cause Blau syndrome, an autoinflammatory disorder involving the joints, skin and eyes. Insights into the mechanism of this association may be gained by a further understanding of where NOD2 is expressed. The objective of this study was to analyze ocular endothelial cells for NOD2 expression. Human ocular tissue was analyzed by immunohistology using anti-NOD2 antisera. RNA isolated from iris, choroid and endothelial cell lines was analyzed by reverse transcription-PCR and real-time quantitative PCR. Gene regulation was studied by treating endothelial cells with TNF-alpha and IFN-gamma. Functional responses were assessed by measuring IL-6 release from endothelial cells treated with muramyl dipeptide (MDP), synthetic lipopeptide (Pam3CSK4) and lipopolysaccharide (LPS). Immunohistological analysis revealed staining of endothelial cells in the uveal tract. NOD2 expression was detected in primary ocular endothelial cell cultures, and levels increased in response to inflammatory cytokines. Endothelial cells from choroid demonstrated enhanced release of IL-6 in response to MDP, and synergy was observed following treatment with MDP and either Pam3CSK4 or LPS. The observations that endothelial cells express NOD2, upregulate NOD2 in response to stimuli known to promote NOD2 expression and show synergistic cytokine responses to MDP and TLR ligands previously shown to be mediated by NOD2 are informative since they may be relevant to pathogenic mechanisms leading to the spectrum of inflammation seen in Blau syndrome.