Alternatively activated macrophages regulate extracellular levels of the hormone placental lactogen via receptor-mediated uptake and transcytosis.

Alternatively activated (M2) macrophages regulate immune responses and tissue remodelling. In many tissues including placenta, M2 express stabilin-1, a multidomain protein that exerts a dual role as a scavenger receptor for acetylated low density lipoprotein (acLDL) and SPARC (secreted protein acidic and rich in cysteine) and as an intracellular cargo carrier for SI-CLP. Using yeast two-hybrid screening, we identified the developmental hormone placental lactogen (PL) as a novel ligand of stabilin-1. In Chinese hamster ovary-stabilin-1 cells and M2, FACS and confocal microscopy demonstrated that stabilin-1 mediates internalization and endosomal sorting of PL. In M2 macrophages, PL was partially degraded in lysosomes; part of PL escaped degradation and was delivered to novel PL+ storage vesicles lacking endosomal/lysosomal markers. During formation, PL+ vesicles underwent transient interaction with the trans-Golgi network (TGN). Upon placement of PL-loaded M2 into PL-free medium, PL was secreted into the supernatant. Leupeptin, an inhibitor of lysosomal hydrolases, reduced PL degradation, enhanced sorting of PL into the TGN/storage vesicle pathway and increased PL secretion. Thus, processing of PL in M2 macrophages occurs either by the classical lysosomal pathway or by a novel TGN-associated trans-secretory pathway. Macrophages isolated from human placental villi efficiently endocytosed PL-FITC and transported it to the storage vesicles. Our data show that extracellular PL levels are determined by uptake, degradation, storage, and release in M2. During pregnancy PL concentration reaches 10 microg/ml in maternal circulation and stays below 0.5 microg/ml in fetal circulation. We propose that stabilin-1-positive macrophages determine the difference in PL levels between maternal and fetal circulation.

Phosphatidylinositide 3-kinase activity is required for stabilin-1-mediated endosomal transport of acLDL.

Stabilin-1 is a type 1 transmembrane receptor specifically expressed by tissue macrophages and sinusoidal endothelial cells. We recently demonstrated that stabilin-1 is involved in the endocytic/recycling pathway and shuttles between the endosomal system and the trans-Golgi network (TGN) in human macrophages. In the present study, we designed a model cell system to study the mechanisms of stabilin-1-mediated endocytosis. Using CHO-K1 cells stably transfected with stabilin-1, we demonstrated that acetylated low-density lipoprotein (acLDL) induces recruitment of stabilin-1 into the endocytic pathway. Stabilin-1 mediates internalisation of acLDL and its delivery to early endosomes, and it is translocated together with its ligand to the late endosomal compartment. Treatment with wortmannin, a specific inhibitor of phosphatidylinositide 3-kinase (PI3K), did not block stabilin-1 mediated internalisation of acLDL as well as its trafficking to early endosomes, whereas it induced enlargement of stabilin-1/acLDL positive endosomes as well as partial dissociation of EEA1 from these structures. The major effect of wortmannin was the abrogation of stabilin-1/acLDL trafficking into the late endocytic pathway. In stabilin-1 positive human type 2 macrophages, wortmannin treatment resulted in formation of both enlarged and small stabilin-1 positive endosomes. This effect, however, was significantly weaker in macrophages as compared to CHO-stabilin-1 cells. Our data indicate that PI3K activity is required for the transfer of stabilin-1 and its ligand acLDL from early to late endosomal compartments.