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...
The matricellular protein SPARC (secreted protein acidic and rich in cysteine) has been implicated in development, differentiation, response to injury, and tumor biology by virtue of its regulation of extracellular matrix production/assembly and its antiadhesive and antiproliferative effects on different cell types. Despite numerous biological activities described for SPARC, cell surface receptors for this protein have not been identified. By phage display and in vitro-binding assays, we now show that SPARC interacts with stabilin-1, a scavenger receptor expressed by tissue macrophages and sinusoidal endothelial cells. The interaction is mediated by the extracellular epidermal growth factor-like region of stabilin-1 containing the sequence FHGTAC. Using FACS analysis and confocal microscopy, we demonstrate that stabilin-1 internalizes and targets SPARC to an endosomal pathway in Chinese hamster ovary cells stably transfected with this receptor. In human macrophages, stabilin-1 expression is required for receptor-mediated endocytosis of SPARC. SPARC was efficiently endocytosed by alternatively activated macrophages stimulated by IL-4 and dexamethasone, but not solely by Th1 or Th2 cytokines. A time course of ligand exposure to alternatively activated macrophages revealed that stabilin-1-mediated endocytosis of SPARC was followed by its targeting for degradation, similar to the targeting of acetylated low density lipoprotein, another stabilin-1 ligand. We propose that alternatively activated macrophages coordinate extracellular matrix remodeling, angiogenesis, and tumor progression via stabilin-1-mediated endocytosis of SPARC and thereby regulate its extracellular concentration.
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.
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