Ole Sonne

Identification of the haemoglobin scavenger receptor.

Intravascular haemolysis is a physiological phenomenon as well as a severe pathological complication when accelerated in various autoimmune, infectious (such as malaria) and inherited (such as sickle cell disease) disorders. Haemoglobin released into plasma is captured by the acute phase protein haptoglobin, which is depleted from plasma during elevated haemolysis. Here we report the identification of the acute phase-regulated and signal-inducing macrophage protein, CD163, as a receptor that scavenges haemoglobin by mediating endocytosis of haptoglobin–haemoglobin complexes. CD163 binds only haptoglobin and haemoglobin in complex, which indicates the exposure of a receptor-binding neoepitope. The receptor–ligand interaction is Ca2+-dependent and of high affinity. Complexes of haemoglobin and multimeric haptoglobin (the 2-2 phenotype) exhibit higher functional affinity for CD163 than do complexes of haemoglobin and dimeric haptoglobin (the 1-1 phenotype). Specific CD163-mediated endocytosis of haptoglobin–haemoglobin complexes is measurable in cells transfected with CD163 complementary DNA and in CD163-expressing myelo-monocytic lymphoma cells.

Insulin binding and hexose transport in rat adipocytes

SummaryInsulin binding, initial velocity of [14C]methylglucose transport, uptake of [14C]deoxy-glucose and conversion of [U-14C]glucose to CO2, glyceride-glycerol and fatty acids were measured at 37 °C in adipocytes from rats of different weights (135–450 g) and therefore with different mean cell volumes (53–389 pl). Insulin binding per cell increased with increasing cell size and binding was 2.3 times higher in the largest cells than in the smallest cells with tracer alone. The difference was largely accounted for by an increase in the apparent affinity. Influx of methylglucose per cell increased with increasing cell size in the absence of insulin and remained constant as a function of cell size in its presence. The effect of insulin ranged from 11 fold in small cells to 3.5 fold in large cells. The rate of conversion of [U-14C]glucose to CO2 and lipids was about half of the rate of methylglucose transport under all conditions. In contrast, the uptake of deoxyglucose in insulin-stimulated cells decreased markedly with increasing cell size. Increasing cell size caused a small decrease in sensitivity which could be explained by a smaller amount of insulin bound per unit surface area. The results show that increasing cell size/animal weight causes changes in insulin binding which may explain changes in sensitivity. In addition, the hexose transport system is modified in a way which is not explained by changes in insulin binding. Finally, changes in deoxyglucose uptake with cell size do not parallel changes in methylglucose transport.

Cellular targets and receptors for interleukin-6 I. In vivo and in vitro uptake of IL-6 in liver and hepatocytes

Abstract. Interleukin‐6 (IL‐6) is a potent stimulator of the hepatic synthesis of acute‐phase proteins. 125I‐labelled IL‐6 disappeared from the blood of rats with an overall half‐time of about 1.5 min; 41% of the injected tracer dose was recovered in the liver by 15 min. The clearance was biphasic. The simultaneous injection of tracer and an excess of unlabelled IL‐6 eliminated the initial rapid phase, and reduced the hepatic uptake to 14%. Light microscopic autoradio‐graphy showed 5% of the grains over non‐hepatocytes, and 80% over hepatocytes, accumulating in areas around the bile canaliculi. Thereafter, degradation products accumulated in the bile. At 4d̀C, isolated rat hepatocytes bound IL‐6 with an apparent Kd of 39 pmol 1‐1 to a uniform class of 4500 receptors per cell with an apparent molar mass of 115–120 kg mol‐1. The HepG2 human hepatocellular cell line bound IL‐6 with an apparent Kd of 21 pmol 1‐1 to a uniform class of 1200 receptors per cell with an apparent molar mass of 155–160 kg mol‐1. At 37d̀C, both cell types endocytosed the bound ligand slowly, and degradation products appeared in the medium after a relatively long lag period (40 min in hepatocytes and 1 h in HepG2 cells).,

Cellular targets and receptors for interleukin-6. II. Characterization of IL-6 binding and receptors in peripheral blood cells and macrophages.

Abstract. Within 15 min, approximately 2–5% of 125I‐labelled interleukin‐6 (IL‐6) injected intravenously into rats was taken up by the spleen. As determined by light microscopic autoradiography, uptake was mainly (60%) accounted for by macrophages in the red pulp. 125I‐IL‐6 binding in rat peritoneal macrophages was quantitatively similar to that in cultured human monocytes and T‐cells. By comparison, IL‐6 binding to polymorphonuclear granulocytes and freshly isolated monocytes was low. Stimulation with antigen, but not with mitogen (PWM), induced receptor presentation in B‐cells, whereas antigen and mitogen downregulated the binding in T‐cells.

Evidence for binding of human pregnancy zone protein-proteinase complex to α2-macroglobulin receptors

125I-labelled human pregnancy zone protein complexed with chymotrypsin was removed from the circulation with a half-time of 2.3 min after intravenous injection in rats. After 6 min about 67% of the label was present in the liver and about 3% was in the spleen, both in male and in female pregnant rats. The half-time of removal was more than 30 min for native pregnancy zone protein. Uptake into other organs, including placentae and feti, was negligible. 30 pM labelled pregnancy zone protein X chymotrypsin was specifically bound to rat hepatocytes and adipocytes and to human fibroblasts and monocyte-derived macrophages at 4 degrees C. Binding was almost completely abolished by a saturating concentration of unlabelled alpha 2-macroglobulin X trypsin. Binding of 15 pM labelled macroglobulin complex was completely abolished by a saturating concentration of pregnancy zone protein X chymotrypsin. In rat hepatocytes, binding of pregnancy zone protein complex was lower than that of alpha 2-macroglobulin complex at low ligand concentrations. Half-maximal receptor occupancy was obtained with about 300 pM pregnancy zone protein complex. Unlabelled alpha 2-macroglobulin or pregnancy zone protein complex failed to accelerate dissociation of the labelled pregnancy zone protein complex under conditions where dissociation of alpha 2-macroglobulin was markedly enhanced. It is concluded that pregnancy zone protein and alpha 2-macroglobulin complexes bind to the same receptors. The quantitative differences may be related to the fact that alpha 2-macroglobulin is a tetramer whereas the functional unit of pregnancy zone protein is probably a dimer.

The mechanism of receptor-mediated degradation of insulin in isolated rat adipocytes: indirect evidence for a non-lysosomal pathway.

Receptor-bound insulin is substrate for a degradation leading to the release of about half the cell-associated [125I]monoiodoinsulin as [125I]monoiodotyrosine. Classical lysosomal inhibitors of the amine type (cloroquine, methylamine and NH+4) only partly inhibited this receptor-mediated degradation. Leupeptin, which is very effective in other systems, was without any effect in the present system. The degradation could not be reduced by lowering the ATP content of the cells. Sulphydryl reagents strongly inhibited the degradation as has also been shown for the cytosolic insulin-specific protease. Microtubules and microfilaments are probably not involved since inhibitors of the cytoskeleton were without marked effects. It is suggested that in the rat adipocyte only a minor part of the receptor-mediated degradation of insulin takes place via the classical endocytotic lysosomal pathway.

Uptake and degradation of insulin and α2-macroglobulin-trypsin complex in rat adipocytes. Evidence for different pathways

The cell association and degradation of insulin and alpha 2-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 microM) reduced both the uptake of alpha 2-macroglobulin X trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2-3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of alpha 2-macroglobulin X trypsin markedly without affecting that of insulin. Leupeptin (100 microM) increased uptake and reduced degradation of alpha 2-macroglobulin X trypsin without affecting insulin. Dansylcadaverine (500 microM) almost abolished uptake and degradation of alpha 2-macroglobulin X trypsin but had little effect on insulin. Moreover, uptake and degradation of alpha 2-macroglobulin X trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin.

A thrombin receptor in resident rat peritoneal macrophages

Resident rat peritoneal macrophages possess 6 x 10(2) high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 x 10(4) low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 degrees C, and after 12 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min-1 at 4 degrees C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radio-activity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor size of Mr 120,000.

Receptor-mediated degradation and internalization of insulin in the adenocarcinoma cell line HT-29 from human colon

In the adenocarcinoma cell line HT-29 receptor-bound insulin is substrate for a proteolytic process leading to the release of about half of the cell-associated [125I]monoiodoinsulin in the form of [125I]iodide and [125I]monoiodotyrosine. Classical lysosomal inhibitors (NH+4, methylamine, leupeptin) did not inhibit this proteolysis. Inhibitors of membrane traffic (chloroquine and monensin) and of metabolism (CN-) inhibited the fractional receptor-mediated degradation. The former led to an increased cell-associated 125I activity whereas the latter reduced the uptake. Sulphydryl reagents inhibited the receptor-mediated degradation. The data are not compatible with a quantitatively major role of lysosomes in the receptor-mediated insulin degradation. However, since the process requires energy it is suggested that the receptor-mediated degradation takes place in vesicles other than secondary lysosomes. The responsible enzyme(s) may belong to the thiol group of proteases. Both insulin and the insulin receptor are internalized as a consequence of incubation of HT-29 cells with insulin.

Receptor-mediated degradation of human growth hormone in rat adipocytes and cultured human lymphocytes (IM-9)

In cultured human lymphocytes (IM-9) and in isolated rat adipocytes human growth hormone is substrate for a receptor-mediated degradation. When the cells are incubated with monoiodinated human growth hormone half of the radioactivity dissociating from the cells is in the form of [125I]monoiodotyrosine. Since IM-9 lymphocytes have no receptor-mediated degradation of insulin, obviously insulin and human growth hormone follow different pathways in this cell type. In the rat adipocyte colchicine and monodansylcadaverine caused quantitatively different uptake and degradation of these 2 ligands suggesting that also in this cell type the pathways are functionally different. The application of different inhibitors suggests that the receptor-mediated degradation of growth hormone in these 2 cell types takes place in an acidified compartment by an energy-requiring process and involving thiol groups.