IntroductionMacrophages are essential elements of innate immunity that orchestrate inflammatory reactions and immune tolerance as well as healing processes and tissue homeostasis. Macrophages perform these functions by the tightly regulated production of cytokines, enzymes, extracellular matrix (ECM) components, and other mediators. Alternatively, macrophages may also bind and internalize these molecules, thereby contributing to their inactivation and degradation. The high specificity and flexibility of this clearance function are based on the expression of multiple endocytic receptors by macrophages. 1 The molecular patterns of macrophage secretory and clearance functions are regulated by the activation status and the polarization of the macrophages involved. [2][3][4] Besides the classic, constitutively operating ER/Golgi secretory pathway, which is regulated primarily on the level of gene expression, macrophages use nonclassic 5 and lysosomal secretory pathways. [6][7][8] The lysosomal secretion route is regulated by specific sorting of newly synthesized products into secretory lysosomes and by stimulus-dependent vesicular/membrane biogenesis. 9 Constitutive sorting of soluble cargo proteins from the Golgi to the endosomal/ lysosomal system is mediated by mannose 6-phosphate receptors CD-MPR and CI-MPR, 2 major sorting receptors in numerous cell types. 10,11 Cell-type-specific mechanisms for the selective delivery of cargo proteins to lysosomes, however, have hitherto remained elusive. Recently, we have presented evidence for the hypothesis that Th2-polarized macrophages use specific, stimulus-dependent mechanisms for protein delivery from the Golgi compartments to the endosomal/lysosomal system, where the selection of the cargo is mediated by stabilin-1. 12 Stabilin-1 is a type 1 transmembrane receptor previously identified by us that shows a unique cell and tissue distribution. 13,14 Stabilin-1 is a marker for alternative macrophage activation 2 ; it is expressed by specialized tissue macrophages in placenta, skin, gut, and pancreas; in cardiac and skeletal muscle; and by sinusoidal endothelial cells in liver, spleen, bone marrow, and lymph nodes. 2,15 In vitro, the expression of stabilin-1 can be induced in monocytederived macrophages by stimulation with interleukin-4 (IL-4) and dexamethasone, but not interferon ␥ (IFN␥). Stabilin-1 functions as an endocytic receptor in endothelial cells and macrophages; its ligand repertoire, however, differs from that of its closest homolog, stabilin-2. 12,16 While stabilin-2 has been shown to be a specialized scavenger receptor of sinusoidal endothelial cells mediating uptake of hyaluronic acid, AGE, and procollagen peptides, the only well-established ligand for stabilin-1 to date is acLDL. 17,18 In addition to endocytosis/recycling, stabilin-1 is involved in trafficking between early/sorting endosomes and the trans-Golgi network 12 in human macrophages. Shuttling of stabilin-1 between For personal use only. on March 26, 2019. by guest www.bloodjournal.org From the bios...

Sentinel lymph node biopsy for several cancers has shown that metastatic tumour cells are preferentially arrested in the lymph node sinuses. To study the molecular components of this sinusoidal trap, gene profiling of lymph node (sinuses) versus tonsil (no sinuses) was performed. Among other groups of molecules, an intriguing gene signature of scavenger and lectin-like receptors was identified. Nine of the 13 genes were preferentially expressed in sinusoidal cells by immunohistochemistry. Using stabilin-2 and monoclonal antibody 3A5 as exclusive endothelial cell (EC) and macrophage (Mvarphi) markers, respectively, lymph node sinusoidal ECs (stabilin-2+, LYVE-1+, DC-SIGNR+, MARCO+, stabilin-1+, MMR+) and sinusoidal Mvarphi (MMR+, DC-SIGN+, sialoadhesin+, CD163+, stabilin-1+ ) showed distinct, but overlapping expression patterns of the signature molecules by double labelling immunofluorescence. The number of stabilin-1+ sinusoidal Mvarphi, however, varied considerably between samples, indicating turnover/differentiation dynamics in this sinusoidal cell population. In the hepatic sinuses, LYVE-1 and CD36 were strongly up-regulated on both sinusoidal ECs and Mvarphi, while DC-SIGNR and DC-SIGN were strongly down-regulated; in contrast to lymph node sinusoidal ECs, MARCO was confined to Mvarphi (Kupffer cells) in the liver sinuses. As Mvarphi are not present in the wall and lumen of splenic sinuses, splenic sinuses expressed a considerably reduced repertoire of scavenger/lectin receptors lacking sialoadhesin, CD36, CD163, and MARCO; in addition, DC-SIGNR was absent from splenic sinusoidal ECs, while DC-SIGN and thrombomodulin were strongly expressed. Interestingly, most of the signature molecules are known to mediate tumour cell adhesion in addition to their functions as scavenger or pattern recognition receptors. This study establishes a gene and tissue database platform to test the hypothesis that additive expression of the lymph node sinus signature genes in sinusoidal ECs and Mvarphi may contribute to selective tumour cell metastasis in lymph nodes and liver including organ-specific mechanisms, such as intraluminal retention or transmigration, while sparing the spleen.

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 μg/ml in maternal circulation and stays below 0.5 μg/ml in fetal circulation. We propose that stabilin-1-positive macrophages determine the difference in PL levels between maternal and fetal circulation.