Stabilin-1, a homeostatic scavenger receptor with multiple functions

The multifunctional scavenger receptor stabilin-1 (STAB1, FEEL-1, CLEVER-1, KIAA0246) was originally identified as the MS-1 antigen, expressed by sinusoidal endothelial cells in human spleen. Extensive histological studies revealed that stabilin-1 is also expressed by tissue macrophages and sinusoidal endothelial cells in the healthy organism; its expression on both macrophages and different subtypes of endothelial cells is induced during chronic inflammation and tumorigenesis. In vitro induction of stabilin-1 in macrophages requires the presence of glucocorticoids. Stabilin-1 is involved in two intracellular trafficking pathways: receptor mediated endocytosis and recycling; and shuttling between the endosomal compartment and trans-Golgi network (TGN). The latter intracellular pathway of stabilin-1 trafficking is mediated by GGAs, clathrin adaptors that interact with the DDSLL motif in the cytoplasmic tail of stabilin-1. When expressed by alternatively activated macrophages, stabilin-1 mediates the uptake and targeting for degradation of acLDL and SPARC, a regulator of tissue remodeling. Likewise, stabilin-1 in macrophages is involved in intracellular sorting and lysosomal delivery of the novel stabilin- 1-interacting chitinase-like protein (SI-CLP). Indirect evidence suggests that stabilin-1 is involved in adhesion and transmigration in various cell types (including tumor cells, leukocytes, and lymphocytes); however, its rapid recycling and scant level of surface expression argue against its universal role in cell adhesion. In summary, stabilin-1 is a homeostatic receptor which links signals from the extracellular environment to intracellular vesicular processes, creating a potential impact on the macrophage secretion profile.     

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.     

Patient Preferences for Biologicals in Psoriasis: Top Priority of Safety for Cardiovascular Patients

Patients with psoriasis are often affected by comorbidities, which largely influence treatment decisions. Here we performed conjoint analysis to assess the impact of comorbidities on preferences of patients with moderate-to-severe psoriasis for outcome (probability of 50% and 90% improvement, time until response, sustainability of success, probability of mild and severe adverse events (AE), probability of ACR 20 response) and process attributes (treatment location, frequency, duration and delivery method) of biologicals. The influence of comorbidities on Relative Importance Scores (RIS) was determined with analysis of variance and multivariate regression. Among the 200 participants completing the study, 22.5% suffered from psoriatic arthritis, 31.5% from arterial hypertension, 15% from cardiovascular disease (myocardial infarction, stroke, coronary artery disease, and/or arterial occlusive disease), 14.5% from diabetes, 11% from hyperlipidemia, 26% from chronic bronchitis or asthma and 12.5% from depression. Participants with psoriatic arthritis attached greater importance to ACR 20 response (RIS = 10.3 vs. 5.0, p<0.001; β = 0.278, p<0.001) and sustainability (RIS = 5.8 vs. 5.0, p = 0.032) but less value to time until response (RIS = 3.4 vs. 4.8, p = 0.045) than those without arthritis. Participants with arterial hypertension were particularly interested in a low risk of mild AE (RIS 9.7 vs. 12.1; p = 0.033) and a short treatment duration (RIS = 8.0 vs. 9.6, p = 0.002). Those with cardiovascular disease worried more about mild AE (RIS = 12.8 vs. 10, p = 0.027; β = 0.170, p = 0.027) and severe AE (RIS = 23.2 vs. 16.2, p = 0.001; β = 0.203, p = 0.007) but cared less about time until response (β = -0.189, p = 0.013), treatment location (β = -0.153, p = 0.049), frequency (β = -0.20, p = 0.008) and delivery method (β = -0.175, p = 0.023) than others. Patients’ concerns should be addressed in-depth when prescribing biologicals to comorbid patients, keeping in mind that TNF antagonists may favourably influence cardiovascular risk.     

Novel function of alternatively activated macrophages: stabilin-1-mediated clearance of SPARC

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.     

Functional significance of non-neuronal acetylcholine in skin epithelia

In recent years, the physiological role of non-neuronal acetylcholine (ACh) and its receptors (AChR) in epidermal physiology has been under intense investigation. However, little is known about the role of the non-neuronal cholinergic system in inflammatory skin diseases. We chose the clinically nicotine-dependent skin disease hidradenitis suppurativa (HS) as model to study the influence of long term nicotine ingestion on epidermal morphology and AChR expression. HS is a chronic inflammatory, disabling disease of unknown pathogenesis emerging from the pilosebaceous unit of the intertriginous areas. In order to correlate our findings to specific nicotine effects, we used the organotypical coculture system (OTC) and raised artificial epidermis in the presence of nicotine. After 12 days in culture control OTC showed a mature epithelium, while nicotine treated OTCs were significantly thicker. Using immunofluorescence analysis, nicotine treated OTCs produced significantly stronger immunoreactivity (IR) for the alpha3, M(3) and M(5) AChR antisera than control. In contrast, the alpha7 nAChR antiserum showed a slightly reduced IR in the granular layer and the alpha9 nAChR IR retracted to the lower suprabasal layers. In HS epidermis we found the strongest IR for all AChR around the follicular infundibulum while in the sinus epithelia it was only weak. In contrast to the nicotine treated OTC, the alpha7 nAChR IR in the hyperplastic HS epidermis was clearly extended to all living layers. Altogether we provide first hints for a causative role of the non-neuronal cholinergic system in the pathogenesis of HS by promoting infundibular epithelial hyperplasia and thus follicular plugging.     

Patient Preferences for Treatment of Psoriasis with Biologicals: A Discrete Choice Experiment

Treatment dissatisfaction and non-adherence are common among patients with psoriasis, partly due to discordance between individual preferences and recommended treatments. However, patients are more satisfied with biologicals than with other treatments. The aim of our study was to assess patient preferences for treatment of psoriasis with biologicals by using computer-based conjoint analysis. Biologicals approved for psoriasis in Germany were decomposed into outcome (probability of 50% and 90% improvement, time until response, sustainability of success, probability of mild and severe adverse events (AE), probability of American College of Rheumatology (ACR) 20 response) and process attributes (treatment location, frequency, duration and delivery method). Impact of sociodemographic and socioeconomic characteristics and disease severity on Relative Importance Scores (RIS) of each attribute was assessed with analyses of variance, post hoc tests, and multivariate regression. Averaged across the cohort of 200 participants with moderate-to-severe psoriasis, preferences were highest for avoiding severe AE (RIS = 17.3), followed by 90% improvement (RIS = 14.0) and avoiding mild AE (RIS = 10.5). Process attributes reached intermediate RIS (8.2–8.8). Men were more concerned about efficacy than women (50% improvement: RIS = 6.9 vs. 9.5, p = 0.008; β = -0.191, p = 0.011 in multivariate models; 90% improvement: RIS = 12.1 vs. 15.4, p = 0.002; β = -0.197, p = 0.009). Older participants judged the probability of 50% and 90% improvement less relevant than younger ones (50% improvement: Pearson’s Correlation (PC) = -0.161, p = 0.022; β = -0.219, p = 0.017; 90% improvement: PC = -0.155, p = 0.028; β = -0.264, p = 0.004) but worried more about severe AE (PC = 0.175, p = 0.013; β = 0.166, p = 0.082). In summary, participants with moderate-to-severe psoriasis were most interested in safety of biologicals, followed by efficacy, but preferences varied with sociodemographic characteristics and working status. Based on this knowledge, physicians should identify preferences of each individual patient during shared decision-making in order to optimize treatment satisfaction, adherence and outcome.     

Expression of stabilin-2, a novel fasciclin-like hyaluronan receptor protein,in murine sinusoidal endothelia,avascular tissues,and at solid/liquid interfaces

Stabilin-2, the hepatic hyaluronan receptor, has recently been cloned by us. Together with stabilin-1, stabilin-2 constitutes a novel family of fasciclin-like hyaluronan receptor homologues. Here, we analyzed expression of stabilin-2 (mStab-2) in a broad array of C57BL/6 mouse organs and tissues. While northern blot analysis showed positive expression of mStab-2 mRNA confined to liver and spleen, immunohistochemistry demonstrated mStab-2 protein expression in the endothelial sinuses of liver, lymph nodes, spleen, and bone marrow, and in specialized structures of eye, heart, brain, and kidney. Expression of mStab-2 was detected in corneal and lens epithelium, in mesenchymal cells of the heart valves, in the ependymal cells lining the ventricles in the brain, and in the prismatic epithelial cells covering the renal papillae. In pathological conditions, such as tumor growth or wound healing processes, mStab-2 was not expressed in the newly formed vasculature or other tissue components. Based on these results, we suggest that mStab-2 might be involved in the clearance of hyaluronan from the lymph or the blood circulation via the network of endothelial sinuses. At the other mStab-2-positive tissues sites that are either avascular and/or demarcate a solid/liquid interface, mStab-2 may serve to maintain tissue integrity by supporting extracellular matrix turnover or it may contribute to maintaining fluidity of bodily liquids by resorption of hyaluronan.     

Clathrin-coated vesicles form a unique net-like structure in liver sinusoidal endothelial cells by assembling along undisrupted microtubules

Liver sinusoidal endothelial cells (LSECs) are highly active professional scavenger cells using clathrin-mediated endocytosis to clear the blood from macromolecular waste products. Using confocal microscopy, we observed a remarkable net-like distribution of clathrin heavy chain (CHC) in LSECs while all other cell types examined including various primary endothelial cells and cell lines showed the well-known punctuate staining pattern representing clathrin-coated vesicles (CCV). The net-like distribution of CHC in LSECs co-localized fully with microtubules, but not with actin. Upon 3D imaging, the net-like distribution of CHC resolved into numerous CCVs organized along the microtubules. The CCVs only partially co-localized with early endosome antigen 1 (EEA1) and adaptor protein 2 (AP-2). Endocytic vesicles containing ligand destined for degradation (FITC-AHGG) were organized along the clathrin/tubulin net-like structures, whereas transferrin-containing recycling vesicles co-localized to a much lower extent. Disruption of the microtubules by nocodazole treatment caused a collapse of the net-like organization of CCVs as well as a profound redistribution of EEA1, AP-2 and FITC-AHGG-containing vesicles, while transferrin internalization and recycling remained unaffected.