The binding of human low-density lipoproteins to the surface of schistosomula of Schistosoma mansoni is inhibited by polyanions and reduces the binding of anti-schistosomal antibodies.

Host molecules such as serum lipoproteins, blood group glycolipids, and histocompatibility antigens may bind to schistosomes and thereby prevent immune recognition of the parasite. This study examines the kinetics of lipoprotein binding, the ability of polyanions to inhibit lipoprotein binding, the binding of anti-schistosomal antibodies to worms that have previously bound low-density lipoprotein (LDL), and the distribution of lipoproteins bound to the parasites. Lipoproteins in human serum (HS) and purified LDL, very low-density lipoprotein (VLDL), and apolipoprotein B (apo B) in defined media were demonstrated on the surface of schistosomula of Schistosoma mansoni by fluorescence and immunoelectron microscopy using a polyclonal goat anti-human apolipoprotein B antibody (anti-apo B). By fluorophotometric microscopy, lipoprotein binding began within 15 minutes and was largely completed within 3 hours of exposure. Lipoprotein binding saturated at 10% HS or 20 micrograms protein/300 microliters of purified LDL. Suramin inhibited LDL binding by 59% in a dose-dependent fashion. In the absence of LDL in the medium, 2 mM suramin dissociated 41% of bound LDL from the worm surface within 15 minutes and 10 mg/ml heparin dissociated 36%. The binding of human anti-schistosomal antibodies to schistosomula was inhibited by bound LDL. By fluorescence microscopy, serum or purified lipoproteins were distributed over the entire surface of the parasite with focal areas of high intensity. Ultrastructurally, reaction product was seen on the outer leaflet of the outer tegumental membrane and in aggregates and surrounding vesicular structures varying in diameter from 13 to 83 nm. These studies demonstrate that lipoproteins bind to the surface of schistosomula. The binding of lipoproteins is partially inhibited by polyanions, reduces the binding of human anti-schistosomal antibodies, and may help the parasite escape the immune response.     

Potential role for scavenger receptors of human monocytes in the killing of Schistosoma mansoni.

Human low-density lipoproteins (LDL) bind specifically and saturably to the surface of the trematode parasite, Schistosoma mansoni, in vitro. Here we have tested whether human monocytes process the bound LDL. Monocytes obtained by leukapheresis generate H2O2, kill schistosomula, and were seen here endocytosing fluorescently labeled human LDL that was bound to the surface of the parasites. Compounds known to inhibit uptake of LDL via the scavenger receptor, namely, acetylated LDL, polyinosinic acid, dextran sulfate, fucoidan, and polyvinyl sulfate, inhibited both endocytosis of LDL and cell-mediated killing. Non-functional analogs of these inhibitors, namely, polycytidylic acid and dextran, did not inhibit either endocytosis or killing. Monocytes obtained from whole blood after venipuncture neither killed the parasite nor endocytosed LDL from the worm surface. Thus, human monocyte killing of schistosomula may involve removal of LDL from the parasite surface via scavenger receptors.     

Human lipoprotein binding to schistosomula of schistosoma mansoni. Displacement by polyanions, parasite antigen masking, and persistence in young larvae.

It was previously shown by the authors that the binding of human low-density lipoprotein (LDL) to the surface of schistosomula inhibits the binding of human anti-schistosomal antibodies and is inhibited by suramin. Here, three questions were considered. 1) Are LDLs bound to schistosomula displaced from the membrane by polyanions? 2) Does bound LDL mask or hide antigens recognized by human anti-schistosomal antibodies? 3) Is LDL, binding capability present when the larvae enter the blood stream? The first question was tested by measuring the percentage of the schistosomular surface membrane covered by LDL after exposure to LDL with or without dextran sulfate or suramin. The bound LDL was visualized with polyclonal goat anti-human apolipoprotein B (anti-apo B) antibodies and peroxidase-conjugated secondary antibodies. After overnight culture in 20 micrograms/300 microliters LDL, 84.0% +/- 0.3% of the parasite surface was covered by LDL reaction product. When the polyanions suramin or dextran sulfate were added to the cultures for 30 minutes, only 59.7% +/- 4.9% of the surface was covered by reaction product, demonstrating that the LDL was partially displaced from the membrane by these compounds. The second question was tested by measuring the binding of human and mouse monoclonal anti-schistosomal antibodies before and after exposure to LDL, with or without partial removal of the bound LDL by suramin. LDL partially inhibited antibody binding in a reversible fashion. The LDL clearly masked parasite antigens, most probably by steric hindrance. However, there may be competitive inhibition of antibody binding by the LDL as well, because human anti-schistosomal antibodies inhibited LDL binding to worms and both human anti-schistosomal antibody and LDL binding to schistosomula were inhibited by suramin. Finally, the third question was tested by quantitative immunofluorescence. The LDL binding capability persisted and nearly doubled by 72 hours after transformation from cercariae. These experiments demonstrated that LDL bound to the surface of schistosomula through the time they enter the blood stream. LDL bound to the parasite surface may help the parasite to evade antibody-dependent cytotoxic reactions by masking parasite antigens.     

Evaluation of monoclonal antibodies to human plasma low density lipoproteins. A requirement for lipids to maintain antigenic structure

Human plasma low density lipoproteins (LDL) are composed of approximately 25% apoproteins and 75% lipids (w/w). Immunochemical properties of LDL were studied using monoclonal antibodies. BALB/c mice were immunized with LDL and the spleen cells from these mice were then fused with a non-immunoglobulin secreting myeloma cell line (F0). The clones producing desirable antibodies were selected to study the antigenic properties of LDL by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay. First, it was found that the maximal binding of 125I-labeled LDL to polyvinyl chloride microtiter dishes was not temperature dependent. The binding affinity was high with a Ka value of approximately 1.9 X 10(10) M-1 while the monoclonal antibodies possessed an affinity to LDL of 5 X 10(8) M-1 which was 2 orders less than the affinity of LDL to the dishes. The former binding, once established, was irreversible as judged by a subsequent incubation with an excess of unlabeled LDL. The latter binding could be displaced by unlabeled LDL. Therefore, the ELISA technique offered a satisfactory approach to study the interaction between LDL and monoclonal antibodies. Removal of lipids from bound LDL by organic extraction resulted in a 50% loss of immunoreactivity, suggesting that the lipids of LDL are important in maintaining the antigenic structure of LDL. Since the apoprotein of LDL also constitutes approximately 40% of the mass (w/w) of very low density lipoproteins (VLDL), the immunoreactivity of VLDL assessed by LDL-monoclonal antibodies was also carried out. Removal of triglycerides from VLDL by lipoprotein lipase resulted in a substantial loss of immunoreactivity as determined by radioimmunoassay. These findings are consistent with the concept that lipids play a role in maintaining the integrity of the antigenic structure of LDL.     

The existence of B/E and E receptors on Hep-G2 cells: a study using colloidal gold- and 125I-labeled lipoproteins

The presence of specific receptors for apolipoprotein B (low-density lipoproteins) and apolipoprotein E (HDL-E) on Hep-G2 cells and human skin fibroblasts was studied by chemical methods and by electron microscopy using a differential gold labeling technique. Fibroblasts bound both types of lipoproteins to one and the same receptor (B/E receptor) as deduced from competition experiments with HDL-E and LDL. Labeled HDL-E, on the other hand, was only partially displaced by cold LDL but was completely displaced by unlabeled HDL-E. Scatchard analysis of lipoprotein binding to Hep-G2 cells revealed an approx 10 times higher binding affinity of apoE-containing lipoproteins as compared to apoB-containing ones. No differences between apoE- or apoB-containing lipoproteins with respect to the morphology of cell binding and intracellular processing were observed. The results are compatible with the concept that Hep-G2 cells possess two kinds of receptors, one specific for apoB- and apoE-containing lipoproteins (B/E receptor) and another specific for apoE only. From these studies we conclude that Hep-G2 cells may serve as a suitable model for studying the lipoprotein metabolism in the liver.     

Studies on the binding of plasma low density lipoproteins to arterial elastin

The mechanism of in vitro complex formation between plasma low density lipoproteins (LDL) and arterial elastin was studied. Rosette formation and decreased binding of the chemically modified LDL suggested that the intact protein moiety of lipoproteins was essential for the transfer of lipids from LDL to elastin. However, subsequent treatment of the elastin-LDL complex with trypsin removed the greater part of the lipoprotein protein but not the transferred cholesterol, indicating that the protein moiety of the lipoprotein did not take part in the retention of lipids on the elastin. In view of the observed effects of pH, ionic strength, various types of detergents and polarity of elastin preparations, it appears that the charged groups of the protein moiety of lipoproteins and the hydrophobicity of elastin proteins may play important parts in the binding of lipoproteins to arterial elastin.     

Modified low-density lipoproteins and high-density lipoproteins. From investigation tools to real in vivo players

It has long been known that the oxidative state of the various plasma lipoproteins modulates platelet aggregability, thereby contributing to atherogenesis. Low-density lipoprotein (LDL), occurring in vivo both in the native and oxidised forms, interacts directly with platelets, by binding to specific receptors. While the identity of the receptors for native LDL and some subfractions of high-density lipoproteins (HDL) remains disputed, apoE-containing HDL(2) binds to LRP8. The nature of these interactions as well as the distinction between candidate receptor proteins was elucidated using covalently modified apolipoproteins, which pointed to the participation of apolipoproteins in high affinity binding. However, the platelet effects initiated by binding of native lipoproteins remain controversial. Some of this ambiguity can be traced to the fact that native LDL inevitably undergoes substantial oxidisation upon modification, including by radiolabelling. The platelet-activating effects provoked by oxidised LDL are irrefutable, but many details remain unknown. The role of CD36 in platelet binding by oxidised LDL is well established, although additional receptors may exist. Much less is known about the interaction of oxidised HDL with platelets, since platelet activation was observed in some, but not all studies. Various frequently applied in vitro oxidation methods produce modified lipoprotein species that may not be relevant in vivo. Based on the reported modifications obtained by in vitro oxidation of LDL, early investigations focused mainly on the formation and the eventual effects of oxidised lipids. More recently, alterations to lipoproteins performed using hypochloric acid and myeloperoxidase redirected the attention to the role of modified apoproteins in triggering platelet responses.     

Scanning electron microscopy of the human low-density lipoprotein interaction with the tegument of Schistosoma mansoni

The interaction between host molecules and Schistosoma mansoni has been regarded as a key feature for parasite survival. In this work, scanning electron microscopy was used to study the interaction of human low-density lipoprotein (LDL) with the tegument of the adult worm of S. mansoni. Worms were incubated in RPMI 1640 containing 10% of LPDS and 40 μg LDL/mL during 30, 60, and 120 min. Control worms were processed in the same way, without LDL. After the incubations, the samples were fixed and processed to scanning electron microscopy. The results demonstrated interaction of the LDL particles with the male parasite tegument. Male and female worms incubated without LDL from 0 (control) to 120 min did not show alterations in the tegument. It was observed a larger number of LDL particles on the dorsal region of male adult worm than others regions (anterior, posterior and gynecophoral canal). The female tegument did not show adherence of LDL. Aggregates on the tegument of the male worm were in greater number and size in the incubation times of 30 and 60 min than 120 min. The comparison between 30 and 120 min of incubation showed that the particles’ size diminished from 2,650–860 nm to 634–363 nm, respectively. Such reduction can be due to the capture and the use of the lipids by the worm. Therefore, the internalization of lipids from LDL by the male worms seems to be a mechanism independent of endocytosis. Differences between males and females suggest lipid transference from male to female through gynecophoral canal.     

Receptor for Fc on the surfaces of schistosomes

Schistosoma mansoni masks its surface with adsorbed host proteins including erythrocyte antigens, immunoglobulins, major histocompatibility complex class I, and beta(2)-microglobulin (beta(2)m), presumably as a means of avoiding host immune responses. How this is accomplished has not been explained. To identify surface receptors for host proteins, we biotinylated the tegument of live S. mansoni adults and mechanically transformed schistosomula and then removed the parasite surface with detergent. Incubation of biotinylated schistosome surface extracts with human immunoglobulin G (IgG) Fc-Sepharose resulted in purification of a 97-kDa protein that was subsequently identified as paramyosin (Pmy), using antiserum specific for recombinant Pmy. Fc also bound recombinant S. mansoni Pmy and native S. japonicum Pmy. Antiserum to Pmy decreased the binding of Pmy to Fc-Sepharose, and no proteins bound after removal of Pmy from extracts. Fluoresceinated human Fc bound to the surface, vestigial penetration glands, and nascent oral cavity of mechanically transformed schistosomula, and rabbit anti-Pmy Fab fragments ablated the binding of Fc to the schistosome surface. Pmy coprecipitated with host IgG from parasite surface extracts, indicating that complexes formed on the parasite surface as well as in vitro. Binding of Pmy to Fc was not inhibited by soluble protein A, suggesting that Pmy does not bind to the region between the CH2 and CH3 domains used by many other Fc-binding proteins. beta(2)m did not bind to the schistosome Fc receptor (Pmy), a finding that contradicts reports from earlier workers but did bind to a heteromultimer of labeled schistosomula surface proteins. This is the first report of the molecular identity of a schistosome Fc receptor; moreover it demonstrates an additional aspect of the unusual and multifunctional properties of Pmy from schistosomes and other parasitic flatworms.     

Evidence for the occurrence of LDL receptors in extracts of schistosomula of Schistosoma mansoni

Schistosomula of Schistosoma mansoni were shown to contain proteins on their surface membranes which bind iodinated human low density lipoproteins (125I-LDL). Treatment of the parasites with trypsin decreased the binding in comparison with untreated controls. Membrane-bound, acetone-insoluble proteins were extracted from the schistosomula with Triton X-100 and the extract in liposome form was incubated with 125I-LDL at room temperature. After incubation a complex was formed between the proteins present in the extract and 125I-LDL, as shown by a filter binding assay. 125I-LDL binding to filters was proportional to the amount of protein in the extract; it was inhibited by unlabelled LDL and VLDL and by EDTA. Binding of 125I-LDL to proteins present in the liposome suspension containing the Triton X-100 extract followed saturation kinetics, indicating the occurrence of receptors for lipoproteins in the extract.     

The interaction of human serum with the surface membrane of schistosomula of Schistosoma mansoni

After contact with human serum, a series of proteins become exposed on the surface membranes of schistosomula of Schistosoma mansoni as revealed by radioiodination of the intact parasites. Among the proteins, a doublet (Mr 45 000) is particularly prominent. These doublet proteins, which are believed to be parasite-derived, become apparent after a very short time of incubation with human serum (10 min or less) and are expressed on the surface membranes after contact with a high molecular weight component of human serum (Mr greater than 80 000). Pretreatment of the parasites with 1.25 mM colchicine or fixation with 2% glutaraldehyde does not prevent the serum-induced expression of the doublet proteins. Extraction of the parasites with chloroform:methanol 2:1 (v/v), however, blocks the human serum effect. Affinity chromatography using immobilized low density lipoproteins (LDL) from human serum shows a tight binding between the 125I-labelled 45 kDa doublet to the LDL. The possible role of the 45 kDa doublet as a receptor for LDL is discussed.     

The exposed carbohydrates of schistosomula of Schistosoma mansoni and their modification during maturation in vivo

Lectins labeled with 125I or conjugated with fluorescein were employed to study the carbohydrates on the surface of different stages of schistosomula of Schistosoma mansoni. Newly transformed schistosomula were shown to bind concanavalin A; the 60 000 and 120 000 dalton agglutinins from Ricinus communis; the fucose-binding protein from Lotus tetragonolobus; wheat germ agglutinin and peanut agglutinin. Soybean agglutinin, Ulex europaeus agglutinin and Dolichos biflorus agglutinin, on the other hand, failed to bind to the schistosomulum surface. The binding of peanut and soybean agglutinin was unaffected by pretreatment of the parasites with neuraminidase. Binding of concanavalin A, the 120 000 dalton agglutinin from Ricinus communis, wheat germ agglutinin and peanut agglutinin to the surface of 5-day schistosomula, recovered from the lungs of mice, was also demonstrated. In each case, however, the level of binding was approximately 70% less than that observed with newly transformed schistosomula and the binding of the fucose-binding protein from L. tetragonolobus practically disappeared. In contrast with newly transformed schistosomula, lung stage schistosomula, pretreated with neuraminidase, displayed a significant increase in the binding of peanut and soybean agglutinin. The results indicate that a significant alteration in the surface carbohydrates of S. mansoni occurs during in vivo maturation of the parasite. This change may contribute to the organism's ability to survive in the vertebrate host.     

Inactivation of rat macrophages by peptides resulting from cleavage of IgG by shistosoma larvae proteases

We have previously reported that IgG molecules bound to the surface Fc receptors of S. mansoni schistosomula were hydrolyzed by parasite enzymes. In this paper, it is shown that the hydrolyzed peptides inhibit macrophage stimulation, assessed by β-glucuronidase release or glucosamine incorporation, and also reduce both phagocytosis of latex beads and IgE-mediated macrophage cytotoxicity against schistosomula.This original process might represent an efficient immunosuppressive mechanism of the parasite to escape the host response.     

A comparative microscopic and biochemical study of the uptake of fluorescent and 125I-labeled lipoproteins by skin fibroblasts, smooth muscle cells, and peritoneal macrophages in culture.

Uptake of low density lipoprotein (LDL) and of acetyl LDL was compared in skin fibroblasts, smooth muscle cells, and peritoneal macrophages with the use of lipoproteins labeled with either 125I or the fluorescent probe 3,3'-dioctadecylindocarbocyanine (DiI). The uptake of DiI-labeled lipoproteins was assessed by quantitative spectrofluorometry and by fluorescence microscopy. The DiI was quantitatively retained by the cells, while the 125I-LDL was degraded and 125I-labeled degradation products were excreted from the cells. In smooth muscle cells and fibroblasts the uptake of LDL was virtually the same whether measured with the use of the DiI or 125I-label (sum of cell-associated and degraded 125I). The labeling of acetyl LDL with DiI enhanced its uptake in peritoneal macrophages by an average of 18%. With the DiI label, lipoprotein uptake (DiI-LDL for smooth muscle cells and skin fibroblasts and DiI-acetyl-LDL for mouse peritoneal macrophages) could be determined after as little as 10 minutes of incubation at 37 C. The pattern of uptake of the DiI-labeled lipoproteins was consistent with binding to specific receptors, because no DiI could be detected in mutant cells without LDL receptors, and uptake was competitively inhibited by addition of excess unlabeled lipoprotein. When the DiI-labeled lipoproteins were removed from the medium, there was a 5-15% loss of DiI from all cell types studied over the first 24 hours. Thereafter, DiI loss from cells was dependent on cell type and culture medium. No further loss of DiI occurred from skin fibroblasts for up to 96 hours of incubation in medium supplemented with either lipoprotein-deficient serum (LPDS) or 10% fetal bovine serum. During this same time period there was a 40-60% loss of DiI from smooth muscle cells and macrophages incubated in medium supplemented with LPDS. Most of the DiI lost from the cells (60-70%) could be recovered in the culture medium but was not the result of cell death, as was indicated by the relatively constant protein concentrations per dish. The loss of DiI was markedly reduced in smooth muscle cells and macrophages when 10% fetal bovine serum was substituted for the LPDS in the culture medium. This suggests that some cells incubated with LPDS undergo changes, perhaps in the plasma membrane, that alter their ability to retain the DiI. In the presence of 10% fetal bovine serum, however, the DiI label is quantitatively retained by all cells tested for up to 96 hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane topology of Borrelia burgdorferi and Treponema pallidum lipoproteins.

A critical issue regarding the molecular architectures of Treponema pallidum and Borrelia burgdorferi, the agents of venereal syphilis and Lyme disease, respectively, concerns the membrane topologies of their major lipoprotein immunogens. A related question is whether these lipid-modified membrane proteins form intramembranous particles during freeze fracture electron microscopy. To address these issues, native borrelial and treponemal lipoproteins were reconstituted into liposomes of diverse composition. The importance of the covalently associated lipids for membrane association of lipoproteins was revealed by the observation that nonlipidated recombinant forms of both B. burgdorferi OspA and the T. pallidum 47-kDa immunogen (Tpp47) showed very weak or no binding to model bilayer vesicles. In contrast to control liposomes reconstituted with bacteriorhodopsin or bovine rhodopsin, two well-characterized transmembrane proteins, none of the lipoprotein-liposomes contained particles when examined by freeze fracture electron microscopy. To extend these findings to prokaryotic lipoproteins with relatively amphiphilic polypeptides, similar experiments were conducted with a recombinant nonlipidated form of Escherichia coli TraT, a lipoprotein which has putative transmembrane domains. The nonlipidated TraT oligomers bound vesicles derived from E. coli lipids but, surprisingly, did not form particles in the freeze-fractured liposomes. These findings support (i) a proposed topology of spirochetal lipoproteins in which the polypeptide is extrinsic to the membrane surface and (ii) the contention that particles visualized in freeze-fractured spirochetal membranes represent poorly characterized transmembrane proteins.     

Surface proteins of Schistosoma mansoni and their expression during morphogenesis

Surface components of Schistosoma mansoni have been identified by lactoperoxidase-catalyzed iodination. Cercariae have a simple labeling pattern in comparison to schis- tosomula. Transformation of cercariae to schistosomula results in the loss of a low molecular weight material which may be the glycocalyx, and the appearance of many more labeled proteins. Mechanical conversion of cercariae to schistosomula requires subsequent incubation at 37 °C for more than 1 h to give the full surface-labeling pattern of schistosomula. The majority of proteins found on schistosomula appear to be present throughout the remaining part of the developmental cycle, although adult male worms had only low levels of these antigens, and female worms had virtually no detectable surface antigens. The low level of expression of schistosome antigen could be caused by adsorbed host antigen, although no evidence for adsorbed host protein was found, or by a reduced level of antigens present on the worm surface. The low level of schistosome antigen could have a role in the resistance of adult worms to the host's immune response.     

Association between coagulation factors VII and X with triglyceride rich lipoproteins.

The association between the concentration of different plasma lipoproteins and plasma factor VII (F VII) was analysed by isolating plasma very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) lipoproteins and assessing their in vitro interaction with F VII by immunoenzyme assay using peroxidase labelled anti-factor VII immunoglobulins to determine whether F VII coagulant activity is prognostic for cardiovascular mortality. F VII bound to triglyceride rich lipoproteins, the fixation being stronger on chylomicrons and VLDL fractions than on LDL fractions. In our experiments HDL did not bind to F VII. The fixation of coagulation factor X (FX) tested by the same method is comparable with that of F VII. The nature of this fixation seemed to arise from hydrophobic interaction as calcium was not necessary and the use of Tween 20 inhibited the interaction. The binding of factors VII and X was increased when lipids were previously treated by phospholipase C and the interaction seemed to be completely dependent on the lipid part of the lipoproteins. Hyrophobic fixation is a possible mechanism of interaction of plasma lipoproteins and F VII and X, and it may be of importance in the covariance of triglyceride concentrations and the activity of vitamin K dependent coagulation factors.     

Complement regulation on the surface of cultured schistosomula and adult worms of Schistosoma mansoni

Cercaria and freshly prepared schistosomula of Schistosoma mansoni are highly sensitive to complement. However, early in their maturation, the schistosomula become resistant to complement killing. This conversion is preceded by a rapid and massive release of several acetabular proteases and of the glycocalyx coat. Thus, shedding of the glycocalyx which is a major immunogen and a strong activator of the alternative pathway of complement permits the parasite to escape immune damage. Mechanically transformed schistosomula, which were cultured in a defined synthetic medium and developed complement resistance, could be converted by proteolysis to complement sensitivity. Trypsin and pronase markedly increased the susceptibility of cultured schistosomula to complement. The trypsin-induced complement sensitivity persisted for at least 19 h without recovery of resistance. Similar treatment with trypsin produced complete killing of adult worms by complement in absence of antibodies. Efficient killing was obtained with normal human serum (NHS), with normal guinea pig serum (GpS), and with C4-depleted HS and C4-deficient GpS indicating that the killing was mediated by the cytolytic alternative pathway of complement. Larger quantities of C3b with intact alpha' chain could be demonstrated on trypsin-treated than on non-treated schistosomula. Antibodies which were raised in rabbits by immunization with the trypsin-released material bound to cultured (non-treated) schistosomula and to adult worms, and induced their killing in GpS and C4-deficient GpS. These results suggest that following release of the glycocalyx, the transforming schistosomula of S. mansoni spontaneously express a complement regulatory protein(s). A similar regulator is postulated to be present on the surface of adult worms. Such regulatory molecules may serve as good targets for immunotherapy, since antibodies directed to them will inhibit their regulatory activity and thus potentiate in vivo the lytic action of complement.     

Low-density lipoprotein endocytosis. II. Influence of the multivalent ligand cationized ferritin on acetylated low-density lipoprotein endocytosis in cultured cells

Interactions of the basic multivalent ligand cationized ferritin (CF) with cultured cells markedly alter their endocytic function. In this study, the influence of CF treatment on the binding, internalization, and degradation of chemically modified (acetylated) low-density lipoproteins (Ac-LDL) was examined in aortic smooth muscle cells (SMC); and in normal and FH mutant LDL receptor-negative human skin fibroblasts, which lack the Ac-LDL (scavenger) receptor; and in vascular endothelial cells, which normally express the receptor. Although CF treatment of all three cell types at 37 degrees C resulted in the induction of Pronasesensitive, high-capacity, high-affinity binding (Kd = 12.0 +/- 2.0 nM at 4 degrees C) of labeled Ac-LDL, which at 37 degrees C was accompanied by significant internalization and degradation, these processes were not receptor-mediated. CF-induced high-affinity binding was inhibited by unlabeled Ac-LDL, fucoidan, carrageenan, and dextran sulfate but was unaffected by native LDL and albumin and only partially inhibited by acetylated albumin. However, analysis of membrane preparations of the cells for "scavenger" receptor protein by solid-phase filtration assay and Western blotting identified the receptor in endothelial cells and in granuloma (positive control) macrophages, but not in either CF-treated or untreated SMC. In addition, studies with both glutaraldehyde-fixed cells and CF bound to culture dishes indicated that Ac-LDL avidly binds to CF. Further, ultrastructural studies using colloidal gold-conjugated Ac-LDL showed Ac-LDL preferentially binding to CF aggregates on the cell surface. Thus, these studies indicate that treatment of cells with CF induces an endocytic process which, although remarkably similar to the scavenger pathway, is mediated by Ac-LDL binding to membrane-associated CF. These observations have implications in terms of mechanisms that might regulate the endocytosis of modified low-density lipoproteins.     

Surface properties of LDL-binding lymphocytes in human peripheral blood.

Surface properties of low density lipoprotein (LDL)-binding lymphocytes were evaluated to determine whether LDL binds with a subpopulation of human peripheral blood lymphocytes (PBL). B- and T-cell rich fractions were prepared from PBL using E-rosette formation or nylon reticulum columns. Binding of FITC-labelled LDL with these cell fractions was determined with a fluorescent microscope and a fluorescence-activated cell sorter (FACS II). The specificity of the binding was evaluated by a dose-dependent inhibition of LDL binding with the addition of unlabelled lipoproteins. In parallel studies, surface properties including E-rosette formation, surface immunoglobulins, and receptors for IgG-Fc, as well as human and mouse C3 were examined. LDL binding lymphocytes were enriched in the B-cell rich fraction, and depleted in the T-cell rich fraction. In addition, FITC-LDL binding lymphocytes were selectively collected by the FACS II. These LDL binding cells restored surface immunoglobulins after incubation in serum-free medium following trypsinization. The majority of lymphocytes stimulated by PHA and PWM in vitro bound with LDL. It is concluded that LDL binds with B cells in fresh human PBL, while it binds with B and T cells in mitogen-stimulated lymphocytes. It is suggested that the selective collection of LDL binding lymphocytes by the FACS II can be applied to the evaluation of cellular interaction of these cells in various immunological reactions.