Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4

Toll-like receptor 4 (TLR4), the principal signaling receptor for lipopolys accharide (LPS) in mammals, requires the binding of MD-2 to its extracellul ar domain for maximal responsiveness. MD-2 contains a leader sequence but l acks a transmembrane domain, and we asked whether it is secreted into the m edium as an active protein. As a source of secreted MD-2 (sMD-2), we used c ulture supernatants from cells stably transduced with epitope-tagged human MD-2. We show that sMD-2 exists as a heterogeneous collection of large disu lfide-linked oligomers formed from stable dimeric subunits and that concent rations of sMD-2 as low as 50 pM enhance the responsiveness of TLR4 reporte r cells to LPS. An MD-2-like activity is also released by monocyte-derived dendritic cells from normal donors. When coexpressed, TLR4 indiscriminately associates in the endoplasmic reticulum/cis Golgi with different-sized oli gomers of MD-2, and excess MD-2 is secreted into the medium. We conclude th at normal and transfected cells secrete a soluble form of MD-2 that binds w ith high affinity to TLR4 and that could play a role in regulating response s to LPS and other pathogen-derived substances in vivo.