Astrid Hammer

Uptake and transport of high‐density lipoprotein (HDL) and HDL‐associated α‐tocopherol by an in vitro blood–brain barrier model

The present study aimed to investigate pathways that contribute to uptake and transcytosis of high‐density lipoproteins (HDLs) and HDL‐associated α‐tocopherol (αTocH) across an in vitro model of the blood–brain barrier (BBB). In primary porcine brain capillary endothelial cells HDL‐associated αTocH was taken up in 10‐fold excess of HDL holoparticles, indicating efficient selective uptake, a pathway mediated by scavenger receptor class B, type I (SR‐BI). SR‐BI was present in caveolae of brain capillary endothelial cells and expressed almost exclusively at the apical membrane. Disruption of caveolae with methyl‐β‐cyclodextrin (CDX) resulted in (mis)sorting of SR‐BI to the basolateral membrane. Immunohistochemistry of porcine brain cryosections revealed SR‐BI expression on brain capillary endothelial cells and presumably astrocytic endfeet. HDL‐associated [14C]αTocH taken up by brain capillary endothelial cells was recovered in sucrose gradient fractions containing the majority of cellular caveolin‐1, the major caveolae‐associated protein. During mass transfer studies using αTocH‐enriched HDL, approximately 50% of cellular αTocH was recovered with the bulk of cellular caveolin‐1 and SR‐BI. Efflux experiments revealed that a substantial amount of cell‐associated [14C]αTocH could be mobilized into the culture medium. In addition, apical‐to‐basolateral transport of HDL holoparticles and HDL‐associated αTocH was saturable. Results from the present study suggest that part of cerebral apolipoprotein A‐I and αTocH originates from plasma HDL transcytosed across the BBB and that caveolae‐located SR‐BI facilitates selective uptake of HDL‐associated αTocH at the BBB.

Fas and Fas-Ligand Are Expressed in the Uteroplacental Unit of First-Trimester Pregnancy

PROBLEM: Fas and Fas‐ligand (FasL) are thought to provide a strategy for reducing graft rejection in immunologically ‘privileged’ tissues by controlling injurious lymphocyte reactions. As the uteroplacental unit is often defined as an immune‐privileged site, we investigated the expression of Fas and FasL in this tissue in the first trimester of pregnancy.

Hypochlorite-modified High Density Lipoprotein, a High Affinity Ligand to Scavenger Receptor Class B, Type I, Impairs High Density Lipoprotein-dependent Selective Lipid Uptake and Reverse Cholesterol Transport

Hypochlorous acid/hypochlorite (HOCl/OCl−), a potent oxidant generated in vivo by the myeloperoxidase-H2O2-chloride system of activated phagocytes, alters the physiological properties of high density lipoprotein (HDL) by generating a proatherogenic lipoprotein particle. On endothelial cells lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) and scavenger receptor class B, type I (SR-BI), act in concert by mediating the holoparticle of and selective cholesteryl ester uptake from HOCl-HDL. We therefore investigated the ligand specificity of HOCl-HDL to SR-BI-overexpressing Chinese hamster ovary cells. Binding of HOCl-HDL was saturable, and the degree of HOCl modification was the determining factor for increased binding affinity to SR-BI. Competition experiments further confirmed that HOCl-HDL binds with increased affinity to the same or overlapping domain(s) of SR-BI as does native HDL. Furthermore, SR-BI-mediated selective HDL-cholesteryl ester association as well as time- and concentration-dependent cholesterol efflux from SR-BI overexpressing Chinese hamster ovary cells were, depending on the degree of HOCl modification of HDL, markedly impaired. The most significant findings of this study were that the presence of very low concentrations of HOCl-HDL severely impaired SR-BI-mediated bidirectional cholesterol flux mediated by native HDL. The colocalization of immunoreactive HOCl-modified epitopes with apolipoprotein A-I along with deposits of lipids in serial sections of human atheroma shown here indicates that the myeloperoxidase-H2O2-halide system contributes to oxidative damage of HDL in vivo.

Amnion Epithelial Cells, in Contrast to Trophoblast Cells, Express All Classical HLA Class I Molecules Together With HLA-G

PROBLEM: The expression of the non‐classical HLA‐G gene has been shown at the protein level on trophoblast‐derived embryonic tissue, like the extravillous cytotrophoblast. However, the presence of HLA‐G on embryoblast‐derived cells is currently controversial. The amnion epithelium is an embryoblast‐derived cell layer covering the amnion cavity and is the main source for the amnion fluid.

Expression of HLA class I molecules in human first trimester and term placenta trophoblast

Abstract.Expression of HLA class I molecules in trophoblast cells from various locations in normal human first trimester and term placenta was investigated by immunohistochemistry with a panel of monoclonal antibodies against the heavy chains or complete HLA class I molecules complexed with β2-microglobulin. These reagents were also employed to distinguish between the products of different HLA class I loci. In addition to previously characterized reagents, a novel monoclonal antibody against HLA-A molecules (TÜ155) was used. Various choriocarcinoma and transfected cell lines served as controls for the specificities of the monoclonal antibodies. Cells in close contact with maternal cells, such as invading trophoblast cells and cells of the basal plate, expressed β2-m micro globulin in association with HLA-G and HLA-C heavy chains. These class I heavy chains may also have been present as isolated molecules, although not in each of the cells. In contrast, cells of the chorion laeve exclusively expressed HLA-G, and not HLA-A, -B, or -C antigens. Our data support the often discussed immune protective function and the regulatory function of the HLA-G molecule, during invasion. In addition, by using monoclonal antibodies HCA2 (anti-HLA-A and -G), HC10 (anti-HLA-B and -C), TÜ149 (anti-HLA-B, -C, and some -A alleles), SFR8-B6 (anti-HLA-Bw6 and some -C), LA45 (some HLA-A and -B), TÜ48 (anti-HLA-Bw4 and some -A), and TÜ155 (anti-HLA-A), we show the presence of HLA-C molecules in all extravillous trophoblast cells of the cell columns and in the basal plate; the trophoblast cells of the chorion laeve lack this antigen. The function of this molecule is not clear, although a protective function against natural killer cell activity in the endometrium is postulated.

Generation, Characterization, and Histochemical Application of Monoclonal Antibodies Selectively Recognizing Oxidatively Modified ApoB-Containing Serum Lipoproteins

To investigate either the role oxidized LDL plays in atherosclerosis or structural changes on the surface of oxidized LDL, monoclonal antibodies (mAbs) are an important tool. After immunizing mice with Cu(2+)-oxidized LDL (oxLDL) and fusion of splenocytes, hybridoma supernatants were screened and cloned. Two mAbs, OB/04 and OB/09 (IgG and IgM), were further characterized. In solid-phase fluorescence immunoassays and Western blot analysis both mAbs reacted with oxLDL, LDL oxidized by a free radical-generating azo compound, or oxVLDL but not with native LDL, acetylated LDL, oxHDL3, azo-oxidized HDL3, or HDL3 modified with malondialdehyde (MDA). In competitive immunoassays with LDL modified by oxidized fatty acid-derived aldehydes, mAb OB/09 strongly reacted with MDA-LDL or MDA-VLDL and LDL modified with 4-hydroxyhexenal followed by 4-hydroxynonenal but not with 4-hydroxyoctenal or hepta-2,4-dienal. mAb OB/04 had a weak affinity for LDL after modification with these aldehydes except for MDA-LDL. LDL modified with arachidonic acid oxidation products (AAOPs) was also recognized by this mAb. However, albumin modified either by the aldehydes applied or by AAOPs did not react with either mAb. Thus, the data indicate that each of the mAbs recognizes a different epitope that is expressed only on apoB-containing lipoproteins upon oxidative modification. An immunostaining with mAb OB/04 was obtained in areas rich in macrophages and in connective tissue of a human atherosclerotic lesion.

Expression of Fas-ligand in first trimester and term human placental villi

The expression of Fas-ligand (FasL) on trophoblast cells is thought to play a role in immune regulation during human pregnancy. However, there are some discrepancies in the published data concerning the cell types expressing FasL in the placental villi. Therefore, we examined the expression of FasL on cryosections of first trimester and term placental tissue with three different anti-sera against FasL, which are in common use. By immunohistochemistry, all three anti-sera principally gave the same staining result. In the first trimester of pregnancy, villous cytotrophoblast cells underlying the syncytium, as well as all extravillous trophoblast cells of cell columns and cell islands, gave a clear, mainly membrane-located staining, whereas the syncytiotrophoblast, which forms the borderline to the maternal blood flow, only gave a spot-like reaction in distinct areas. The same result was obtained with term placental villi; however, in this tissue, the staining of the villous cytotrophoblast cells was less pronounced. From our results, we suggest that in placental villi, an important role of FasL in immune regulation is not very conclusive because this molecule is mainly expressed on trophoblast with no access to maternal blood or tissue. This is in contrast to the uterine part of the placenta, where FasL expressing trophoblast cells are in close contact with apoptotic maternal leukocytes.

Uptake of Lipoprotein-Associated α-Tocopherol by Primary Porcine Brain Capillary Endothelial Cells

Abstract: From the severe neurological syndromes resulting from vitamin E deficiency, it is evident that an adequate supply of the brain with α‐tocopherol (αTocH), the biologically most active member of the vitamin E family, is of utmost importance. However, uptake mechanisms of αTocH in cells constituting the blood‐brain barrier are obscure. Therefore, we studied the interaction of low (LDL) and high (HDL) density lipoproteins (the major carriers of αTocH in the circulation) with monolayers of primary porcine brain capillary endothelial cells (pBCECs) and compared the ability of these two lipoprotein classes to transfer lipoprotein‐associated αTocH to pBCECs. With regard to potential binding proteins, we could identify the presence of the LDL receptor and a putative HDL3 binding protein with an apparent molecular mass of 100 kDa. At 4°C, pBCECs bound LDL with high affinity (KD = 6 nM) and apolipoprotein E‐free HDL3 with low affinity (98 nM). The binding capacity was 20,000 (LDL) and 200,000 (HDL3) lipoprotein particles per cell. αTocH uptake was approximately threefold higher from HDL3 than from LDL when [14C]αTocH‐labeled lipoprotein preparations were used. The majority of HDL3‐associated αTocH was taken up in a lipoprotein particle‐independent manner, exceeding HDL3 holoparticle uptake 8‐ to 20‐fold. This uptake route is less important for LDL‐associated αTocH (αTocH uptake ∼1.5‐fold higher than holoparticle uptake). In line with tracer experiments, mass transfer studies with unlabeled lipoproteins revealed that αTocH uptake from HDL3 was almost fivefold more efficient than from LDL. Biodiscrimination studies indicated that uptake efficacy for the eight different stereoisomers of synthetic αTocH is nearly identical. Our findings indicate that HDL could play a major role in supplying the central nervous system with αTocH in vivo.

Hypochlorite-modified albumin colocalizes with RAGE in the artery wall and promotes MCP-1 expression via the RAGE-Erk1/2 MAP-kinase pathway

Signal transduction via the endothelial receptor for advanced glycation end products (RAGE) plays a key role in vascular inflammation. Recent observations have shown that the myeloperoxidase‐H2O2‐chloride system of activated phagocytes is highly up‐regulated under inflammatory conditions where hypochlorous acid (HOCl) is formed as the major oxidant. Albumin, an in vivo carrier for myeloperoxi‐dase is highly vulnerable to oxidation and a major representative of circulating advanced oxidized proteins during inflammatory diseases. Immunohistochem‐ical studies performed in the present study revealed marked colocalization of HOCl‐modified epitopes with RAGE and albumin in sections of human atheroma, mainly at the endothelial lining. We show that albumin modified with physiologically relevant concentrations of HOCl, added as reagent or generated by the myelo‐peroxidase‐H2O2‐chloride system, is a high affinity li‐gand for RAGE. Albumin, modified by HOCl in the absence of free amino acids/carbohydrates/lipids to exclude formation of AGE‐like structures, induced a rapid, RAGE‐dependent activation of extracellular signal‐regulated kinase 1/2 and up‐regulation of the proin‐flammatory mediator monocyte chemoattractant pro‐tein‐1. Cellular activation could be blocked either by a specific polyclonal anti‐RAGE IgG and/or a specific mitogen‐activated protein‐kinase kinase inhibitor. The present study demonstrates that HOCl‐modified albumin acts as a ligand for RAGE and promotes RAGEmediated inflammatory complications.—Marsche, G., Semlitsch, M., Hammer, A., Frank, S., Weigle, B., Demling, N., Schmidt, K., Windischhofer, W., Waeg, G., Sattler, W., Malle, E. Hypochlorite‐modified albumin colocalizes with RAGE in the artery wall and promotes MCP‐1 expression via the RAGE‐Erk1/2 MAP‐kinase pathway. FASEB J. 21, 1145–1152 (2007)

Differential Phenotypic Properties of Human Peripheral Blood CD56dim+ and CD56bright+ Natural Killer Cell Subpopulations

We investigated the presence of integrins and various other cell surface markers on the surface of freshly isolated CD56dim+ and CD56bright+ NK cells, the latter comprising a mean of 6.5% of total peripheral blood (PB) CD3-CD56+ NK cells. This small subpopulation stained more intensively for CD2, CD11c, CD15s, CD44, CD49e, CD55, CD62L, HLA-DR, and GZS-1, whereas there was no expression of CD49f in contrast to CD56dim+ cells. The myeloid antigen CDw65 was present on 11% of CD56bright+ cells, other myeloid markers were not found. 28% of CD56bright+ cells were positive for CD45R0. These results support the notion that CD56bright+ NK cells, based on their different marker profile, may possess different functional and biodistributional properties and--due to the signal-transducing capabilities of several adhesion molecules--differential patterns of stimulatability compared to the majority of PB-NK cells.