The macrophage mannose receptor promotes uptake of ADAMTS13 by dendritic cells

Abstract

ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation by cleaving ultra-large VWF multimers on the surfaces of endothelial cells. Autoantibodies directed against ADAMTS13 prohibit the processing of VWF multimers, initiating a rare and lifethreatening disorder called acquired thrombotic thrombocytopenic purpura. The formation of autoantibodies depends on the activation of CD4 ؉ T cells. This process requires immune recognition, endocytosis, and subsequent processing of ADAMTS13 into peptides that are presented on MHC class II molecules to CD4 ؉ T cells by dendritic cells (DCs). In the present study, we investigated endocytosis of recombinant ADAMTS13 by immature monocyte-derived DCs using flow cytometry and confocal microscopy. After incubation of fluorescently labeled ADAMTS13 with DCs, significant uptake of ADAMTS13 was observed. Endocytosis of ADAMTS13 was completely blocked by the addition of EGTA and mannan. IntroductionAPCs continuously sample antigens from their environment and, after their processing, load antigen-derived peptides on MHC class I or II molecules for presentation to CD8 ϩ or CD4 ϩ T cells, respectively. To appropriately respond to incoming pathogens, APCs are equipped with a large diversity of cellsurface receptors, so-called pattern recognition receptors that recognize specific pathogen-associated molecular patterns. 1 This provides APCs such as dendritic cells (DCs) with the capacity to recognize a wide diversity of foreign pathogens. After encountering a pathogen, DCs undergo maturation and migrate to the draining lymph nodes, where they encounter and prime naive T cells. This mechanism ultimately generates a humoral response that is capable of rapidly neutralizing re-entering pathogens. It is well established that, in addition to recognizing pathogen-associated molecular patterns, pattern recognition receptors can also interact with molecular patterns present on endogenous proteins. 2 This results in the continuous presentation of "self-derived" peptides to CD4 ϩ T cells. 3 The efficient removal of potentially self-reactive T cells in the thymus prevents the stimulation of CD4 ϩ T cells by self-antigens presented on MHC class II molecules. Nevertheless, autoimmunity can develop as a result of activation of low-affinity CD4 ϩ T cells that have not been efficiently eliminated from the repertoire in the thymus. 4 In addition, shared T-cell epitopes between pathogen-encoded and self-antigens are a potential source for cross-reactive T cells. 5 The autoimmune disorder thrombotic thrombocytopenic purpura (TTP) results from the development of autoantibodies directed toward the metalloprotease ADAMTS13. 6 Several studies have shown that these Abs target an exposed surface in the spacer domain,which mediates binding of ADAMTS13 to its substrate, VWF. 6-13 Lack of cleavage of ultra-large VWF multimers by ADAMTS13 results in the accumulation of blood platelets at sites of vascular perturbation. 14 This promotes microvascular obstruction, resulting in low platelet...

The class I scavenger receptor CD163 promotes internalization of ADAMTS13 by macrophages

“…30 We previously showed that the MMR, a mannan-sensitive CLR, mediates internalization of ADAMTS13 by DCs. 16 Although mannan significantly reduced ADAMTS13 uptake by macrophages, ADAMTS13 endocytosis seems to be only partly dependent on this C-type lectin, because its uptake by macrophages is not influenced by the presence of the blocking anti-MR antibody clone 15.2 or by the addition of D-mannose or GlcNAc, which interfere with binding of ligands to mannan-sensitive CLRs. 43 These data suggest that MMR does not play a major role in ADAMTS13 internalization by macrophages.…”

“…We have previously shown that the MMR mediates the uptake of ADAMTS13 by DCs. 16 MMR is predominantly internalized into early endosomes 16 and, after release of its ligand, recycles to the plasma membrane. 31 Confocal microscopy revealed that part of the endocytosed ADAMTS13-AF488 co-localized with the MMR (Figure 2C), suggesting that ADAMTS13 and MMR localize to the same endocytic compartment.…”

“…Previously, we showed that recombinant ADAMTS13 (rADAMTS13) is endocytosed by DCs through a receptor-mediated pathway. 16 To analyze the mechanism for uptake of rADAMTS13, macrophages were preincubated for 20 minutes at 37°C with dynasore (80 mM) before the addition of ADAMTS13-AF488 ( Figure 1D). Dynasore blocks the GTPases dynamin-1 and dynamin-2, which are involved in membrane fission, thereby promoting clathrin-mediated endocytosis as well as other endocytic processes.…”

“…Subsequent purification was performed by using the polyhistadine tag at the C terminus of the protein using theÄKTA purifier system (GE Healthcare) and an Ni-NTA column (GE Healthcare) essentially as described. 16,26 The purified protein was collected and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by silver staining or immunoblotting. For flow cytometry and confocal microscopy, ADAMTS13 was directly labeled with Alexa Fluor 488 (AF488) by using the Alexa Fluor 488 Microscale Labeling Kit according to the manufacturer's protocol (Thermo Fisher Scientific).…”

“…[13][14][15] We have previously shown that ADAMTS13 is endocytosed by antigen-presenting cells such as dendritic cells (DCs) by the macrophage mannose receptor (MMR). 16 After its intracellular processing, a restricted set of ADAMTS13-derived peptides is presented on major histocompatibility complex class II for presentation to CD4…”