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Identification of the 80-kDa LPS-binding protein (LMP80) as decay-accelerating factor (DAF, CD55)
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Identi¢cation of the 80-kDa LPS-binding protein (LMP80) as decay-accelerating factor (DAF, CD55) Volker T. El-Samalouti a , Jens Schletter b , Ines Chyla a , Arnd Lentschat a , Uwe Mamat a , Lore Brade a , Hans-Dieter Flad a , Artur J. Ulmer a; *, Lutz Hamann a
a
Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany b Cardiogene, Gentherapeutische Systeme AG, D-40699 Erkrath, Germany Received 15 October 1998; received in revised form 2 December 1998; accepted 3 December 1998
Abstract The activation of immunocompetent cells by lipopolysaccharide (LPS) during severe Gram-negative infections is responsible for the pathophysiological reactions, possibly resulting in the clinical picture of sepsis. Monocytes recognize LPS mainly through the LPS receptor CD14, however, other cellular binding structures have been assumed to exist. In previous studies, we have described an 80-kDa LPS-binding membrane protein (LMP80), which is present on human monocytes as well as endothelial cells. Here we demonstrate that LMP80 is widely distributed and that it formes complexes together with LPS and sCD14. Furthermore, we report on the biochemical purification of LMP80 and its identification as decay-accelerating factor, CD55, by amino acid sequencing and cloning techniques. Our results imply a new feature of CD55 as a molecule which interacts with LPS/sCD14 complexes. However, the involvement of CD55 in LPS-induced signaling remains to be elucidated. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : CD55 ; Decay-accelerating factor ; Endotoxin; Lipopolysaccharide ; Lipopolysaccharide-binding protein; LMP80
1. Introduction Lipopolysaccharide (LPS, endotoxin), the major component of the outer lea£et of Gram-negative bacteria, is a potent inducer of an in£ammatory response [1,2]. LPS consists of a polysaccharide region * Corresponding author. Tel.: +49 (4537) 188448; Fax: +49 (4537) 188435; E-mail: [email protected] Abbreviations: DAF, decay-accelerating factor; LBP, lipopolysaccharide-binding protein; LMP80, 80-kDa LPS-binding membrane protein; NHS, normal human serum; PVA, polyvinyl alcohol
and a lipid part, called lipid A, which carries all endotoxic properties of the molecule [3,4]. LPS can lead to stimulation of immunocompetent cells, in particular monocytes/macrophages, resulting in the release of immune mediators such as the interleukins (IL) IL-1, IL-6, IL-8, IL-12 and tumor necrosis factor K (TNFK) [5,6]. The activation of cells through LPS requires the interaction of LPS with speci¢c cell surface molecules, capable of initiating transmembrane signaling. It is undisputed that the main binding site of LPS on monocytes/macrophages and granulocytes is the 55-kDa, glycosylphosphatidylinositol (GPI)-anchored CD14 molecule [7,8]. LPS bind-
0928-8244 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 9 8 ) 0 0 1 4 5 - X
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ing to membrane-linked CD14 (mCD14) as well as to the soluble form of CD14 (sCD14) is mediated by LPS-binding protein (LBP), an acute phase reactant present in serum [9,10]. However, there are many indications suggesting the presence of an accessory molecule beside or downstream of mCD14 involved in LPS signaling. Because it lacks a transmembrane domain, mCD14 alone seems to be incapable of transmitting the signal through the membrane. Furthermore, monocyte activation with high LPS doses cannot be blocked by anti-CD14 antibodies [11,12]. Therefore, it is assumed that CD14 mediates the recognition of low LPS doses, whereas high doses of LPS interact directly with another molecule. Additionally, cells of non-hematopoietic origin, such as endothelial cells or ¢broblasts, are responsive to LPS/sCD14 complexes [13^15], which indicates the existence of at least one target structure for the speci¢c recognition of sCD14/LPS complexes on these cells. Several molecules have been described that bind LPS, but an involvement in signal transduction events was only shown for the L-integrins [16^18] and recently for the human Toll-like receptor-2 [19]. In the past, we described a possible candidate for an additional LPS receptor connected to CD14, i.e. an 80-kDa LPS-binding membrane protein (LMP80), which binds LPS in a ligand blotting assay only in the presence of sCD14 and LBP [20]. We found LMP80 expressed on hematopoietic cells (monocytes, erythrocytes) as well as on non-hematopoietic cells (endothelial cells) [20,21]. Furthermore, we con¢rmed the interaction of LPS with LMP80 in its natural membranous environment by immuno-coprecipitation [21]. For this technique, cell membranes were incubated with LPS, solubilized by mild detergent treatment and the complexes of LPS and LMP80 were coprecipitated by the use of anti-LPS monoclonal antibodies (mAbs), indicating an association between the two molecules. The results presented in this paper provide proof that LMP80 is identical to CD55, the decay-accelerating factor (DAF). CD55 is a complement regulatory molecule, present on almost all cells that are in contact with the blood stream [22,23]. It protects the cells from autologous complement lysis by accelerating the decay of the C3 convertases of the classical and the alternative complement pathways [24].
Although CD55, like mCD14, is linked to the cell membrane by a GPI anchor in the cell membrane, it has repeatedly been shown to be involved in signal transduction on T-cells as well as on monocytes [25^ 28]. A functional involvement of CD55 in LPS induced signaling, however, remains to be investigated in further studies.
2. Materials and methods 2.1. Reagents and antibodies Unless otherwise indicated all ¢ne chemicals were purchased from Sigma (Deisenhofen, Germany), Serva (Heidelberg, Germany), Merck (Darmstadt, Germany), Bio-Rad (Munich, Germany), Gibco-BRL (Eggenstein, Germany), or Boehringer Mannheim (Mannheim, Germany). The anti-CD55 antibody IA10 was from Pharmingen (San Diego, CA, USA). Anti-CD14 antibodies were from Immunotech (IOM2), Becton Dickinson (Leu-M3) or Biometec (ROMO-1). MEM-18 [29] was kindly provided by Dr. V. Horejsi (Institute of Molecular Genetics, Prague, Czech Republic). Anti-lipid A mAb A6, which recognizes the phosphorylated carbohydrate backbone of lipid A independently of the acylation pattern, was used as hybridoma serum-free supernatant [30]. Peroxidase-conjugated or FITC-labeled goat antimouse immunoglobulin G was purchased from Dianova (Hamburg, Germany). IgG 2a and IgG 2b isotype control antibodies were purchased from Sigma. Normal human sera were obtained from healthy volunteers. LPS from Escherichia coli Re mutant F515 was prepared by the hot phenol-water procedure, puri¢ed by repeated ultracentrifugation, and converted into the sodium salt form by electrodialysis [31]. Lipid A from E. coli Re mutant F515 was prepared by treating LPS with acetate bu¡er [32]. All LPS and lipid A preparations were solubilized in pyrogen-free distilled water and stored in aliquots at 1 mg ml31 and 4³C. Oligonucleotides were synthesized by TIB Molbiol (Berlin, Germany) or MWG (Munich, Germany). Cloning vectors pCR II and pCEP4 were obtained from Invitrogen (Leek, The Netherlands).
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2.2. Cell separation Human blood cells from healthy donors were separated by density gradient centrifugation of heparinized blood with Ficoll-Paque [33] into two fractions, mononuclear cells and erythrocytes/ granulocytes. For erythrocyte preparation the pellet of the density gradient centrifugation was harvested without the granulocyte layer, washed three times with RPMI 1640 and resuspended in an equal volume of RPMI 1640. The cells were applied to a dextran cushion (3% dextran in 150 mM NaCl) and sedimented at room temperature for 18 min. The erythrocytes were collected and washed two times with PBS. For the isolation of granulocytes, the overlayer of the erythrocyte pellet was applied to a PVA cushion (1% PVA in 0.9% NaCl) and sedimented at room temperature for 20 min. The pellet was collected and erythrocytes were lysed by incubation in 5 ml distilled water. After 45 s 5 ml of 2UPBS was added. Monocytes were isolated by counter£ow elutriation of mononuclear cells as described [20], T-lymphocytes, endothelial cells and smooth muscle cells were prepared as described elsewhere [34^36]. 2.3. Cell culture The monocytic cell line Mono-Mac-6 was kindly provided by Dr. H.W.L. Ziegler-Heitbrock (Institute of Immunology, University of Munich, Munich, Germany). The monocytic cell lines THP-1 and HL-60 were from the American Tissue Culture Collection. All monocytic cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, penicillin (100 U ml31 ), and streptomycin (100 Wg ml31 ). CHO-K1 cells from the DSMZ (Braunschweig, Germany) were cultured in Ham's F12 medium supplemented with 10% fetal calf serum, penicillin (100 U ml31 ), and streptomycin (100 Wg ml31 ). Transfectants were cultured in the presence of 400 Wg ml31 hygromycin. All cultures were maintained in a humidi¢ed incubator at 37³C and 5% CO2 . 2.4. Cell membrane preparation Packed erythrocytes (10 ml) were resuspended in 260 ml hypotonic lysis bu¡er (10 mM Tris-HCl, pH
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7.4) and shaken for 15 min on ice. The lysate was centrifuged at 18 000Ug, 15 min, 4³C, the pellet was washed once with hypotonic lysis bu¡er and twice with distilled water. The membranes were pelleted by a ¢nal centrifugation step (50 000Ug, 35 min, 4³C) and stored at 320³C. Membranes of all other cells and human cell lines were prepared as described elsewhere [20]. CHO cells were harvested after washing monolayers with PBS by incubation with Cell Dissociation Solution (Sigma, Deisenhofen). 50U106 cells were resuspended in 1 ml ice cold lysis bu¡er (10 mM Tris-HCl, pH 7.4 with 1 mM phenylmethylsulfonyl £uoride (PMSF), 2 mM EDTA) and subsequently sonicated on ice in a Branson soni¢er 250 (10 strokes, intensity 1.1, duty cycle 80%). After removal of debris by centrifugation (400Ug, 15 min, 4³C), enriched cell membrane fractions were obtained by high speed centrifugation (36 000Ug, 30 min, 4³C), and aliquots were stored at 320³C. 2.5. Electrophoresis, immunoblotting and ligand blotting assay Probes were subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (LMP80 and CD14 detection) or under non-reducing conditions (CD55 detection) on a 10% gel, followed by blotting of the proteins onto nitrocellulose membranes using a semidry blotting apparatus for 2 h at 2.5 mA cm32 . Nitrocellulose membranes were blocked for 2 h at room temperature with PBS containing 5% non-fat dry milk (PBS/M). The ligand blotting assay for the detection of LMP80 was performed as described elsewhere with some modi¢cations [20]. Brie£y, blocked membranes were incubated for 1.5 h with 0.5 Wg ml31 lipid A in PBS/M-0.1% L-octylglycoside in the presence or absence of 3% NHS. After four washes in PBS containing 0.05% Tween 20 (PBS/T), bound lipid A was detected by anti-lipid A mAb A6. For the detection of CD55, blocked membranes were incubated with anti-CD55 mAb IA10 (1:5000) in PBS/M overnight at 4³C. On the next day, immunoblots were washed four times in PBS/T, incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse mAb in PBS/
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M, and developed with the ECL system (Amersham Buchler, Braunschweig, Germany). 2.6. Puri¢cation of LMP80 Erythrocyte membranes containing 40 mg protein were solubilized in 20 ml solubilization bu¡er (2.5% L-octylglycoside, 1 mM PMSF, 1 mM EDTA) for 30 min at 4³C. After centrifugation (100 000Ug, 30 min, 4³C) supernatants were supplemented to a ¢nal volume of 50 ml with 5% glycerol, 1 g L-octylglycoside, urea (to 2 M), PMSF (to 1 mM), ampholytes (40% Biolyte 3-10 and 40% Biolyte 4-6). Preparative isoelectric focusing (IEF) was performed at 4³C and 12 W in a Rotofer cell (Bio-Rad, Munich, Germany) for 5 h. The LMP80-containing fractions as detected by ligand blotting assay were pooled, washed with equilibration bu¡er (50 mM NaH2 PO4 , 500 mM NaCl, 0.1 mM CaCl2 , 0.1 mM MgCl2 , 1% L-octylglycoside, pH 7), and concentrated in a Centricon-50 (Amicon, Beverly, MA, USA). LMP80 was further enriched by a¤nity chromatography on a column of wheat germ agglutinin (WGA) coupled to agarose. The column was loaded with the concentrated fractions from IEF (2 ml h31 ), washed with equilibration bu¡er (10 ml at 4 ml h31 ), and eluted with elution bu¡er (equilibration bu¡er with 0.5 M WGA). 0.5ml fractions were collected, protein bands were detected by silver staining, and the presence of LMP80 was checked using the ligand blotting assay. LMP80containing fractions were then pooled, concentrated in a Centricon-50 (Amicon, Beverly, MA, USA), and further puri¢ed on a 9% preparative SDS-PAGE in a Model 491 Prep Cell (Bio-Rad, Munich, Germany) at 40 mA. 2.5-ml fractions were collected and the purity of LMP80 was estimated to be greater than 95% by determination on a silver-stained gel. 2.7. Amino acid sequencing Amino acid sequencing of LMP80 was performed by Eurogentec (Seraing, Belgium). 2.8. Generation of CD55 cDNA and cell transfection Monocyte mRNA was isolated from 2U106 cells using oligo-dT-coated magnetic beads (Dynal, Hamburg, Germany), according to the manufacturer's in-
structions, and cDNA was prepared by subsequent reverse transcription as described [37]. Ampli¢cation of CD55 cDNA was carried out using an automatic DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT, USA), and the oligonucleotide pair (5P ATA TGG ATC CAC CAT GAC CGT CGC GCG GCC GAG C+5P ATA TTC TAG ACT AAG AAA CTA GGA ACA GTC TGT ATA CTT) used was designed to introduce a BamHI site, a Kozak sequence, and an XbaI site into the product. The PCR conditions were: 20 cycles, annealing temperature 60³C. The PCR product was cloned in E. coli DH5K employing the TA vector pCR II (Invitrogen). Positive clones were sequenced with the USB Sequenase 2.0 Kit (Amersham Buchler, Braunschweig, Germany) and subcloned into the KpnI and XbaI sites of the vector pCEP4 (pCEP4-CD55). Control sequencing of the ¢nal construct was carried out at MWG (Ebersbach, Germany). CHO cells were transfected with pCEP4 (CHOpCEP4) or pCEP4-CD55 (CHO-CD55) by using Fugene1 (Boehringer Mannheim) as described in the manufacturer's protocol, and selection of positive transfectants was performed after 48 h with hygromycin (400 Wg ml31 ). CD55-positive cells were enriched ( s 80%) by cell sorting with a FACSstar (Becton Dickinson) using anti-CD55 mAbs.
3. Results 3.1. Expression of LMP80 in di¡erent cell types In previous studies we have found that LMP80 is expressed on monocytic cells as well as on endothelial cells and erythrocytes. Furthermore, we checked several other myeloid and non-myeloid cells and cell lines for their LMP80 expression. We analyzed cell membrane preparations containing 30 Wg of total protein by the ligand blotting assay (Fig. 1). We found that LMP80 is present in all tested cells but there are striking di¡erences in the protein quantity (Fig. 1). The amount of LMP80 was similar in monocytes, T-lymphocytes, erythrocytes, granulocytes, endothelial cells, and in the cell lines MonoMac-6 and U937. Smooth muscle cells and the promyeloid cell lines THP-1 and HL-60 showed only weak LMP80 expression. It should be noted that
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Fig. 1. Expression of LMP80. Cell membrane preparations containing 30 Wg of total protein were separated by SDS-PAGE and blotted. Lanes were split, incubated with lipid A (0.5 Wg ml31 ) in the presence (+) or in the absence (3) of normal human serum (NHS) and bound lipid A was detected by anti-lipid A mAb. LMP80 was principally expressed on all investigated cells types, but THP-1 and HL-60 cells contained only minute amounts of LPM80.
the ligand blotting assay was performed under reducing conditions and, therefore, binding of lipid A to CD14 on CD14-positive cells is not observed. 3.2. Dose-dependent binding of lipid A to LMP80 In further studies we analyzed the in£uence of lipid A concentration on the binding of lipid A to LMP80. We found that minimal concentrations (1 ng ml31 ) of lipid A are su¤cient to bind to LMP80 in a serum-dependent manner (Fig. 2). When high concentrations of lipid A were used, serum was not required for the mediation of the binding of lipid A to LMP80.
Fig. 2. Dose-dependent binding of lipid A to LMP80. MonoMac-6 membrane preparations containing 30 Wg of total protein were separated by SDS-PAGE and blotted. Lanes were split and incubated with various lipid A concentrations in the presence (+) or in the absence (3) of NHS. Bound lipid A was detected by anti-lipid A mAb. Binding of lipid A to LMP80 was detectable even in very low lipid A concentrations (1 ng ml31 ). High LPS doses ( s 10 Wg ml31 ) were capable of binding to LPM80 independently of sCD14.
3.3. Presence of sCD14 in the lipid A/LMP80 complex sCD14 is required to mediate together with LBP the binding of low amounts of lipid A to LMP80 [20]. We therefore analyzed whether sCD14 is physically present in the LMP80/lipid A complex. We incubated the electrophoretically separated and blotted proteins with lipid A in the presence or absence of serum. Instead of lipid A detection, we incubated those membranes with di¡erent anti-CD14 antibodies. With the anti-CD14 mAbs ROMO-1, IOM2, and
Fig. 3. Detection of sCD14 in the LMP80/lipid A complex. Mono-Mac-6 membrane preparations containing 30 Wg of total protein were separated by SDS-PAGE and blotted. Lanes were split and incubated with various lipid A concentrations in the presence (+) or in the absence (3) of NHS. Bound sCD14 was detected by anti-CD14 mAbs. The presence of sCD14 complexed to LMP80/lipid A was detected with the mAbs IOM2, Leu-M3 and Romo-1, whereas the clone MEM-18 failed in sCD14 detection.
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Fig. 4. Preparative IEF for LMP80 enrichment. Erythrocyte membranes were solubilized with L-octylglycoside and separated by isoelectric focusing in a Rotofor cell. The obtained fractions were analyzed for their protein concentration and their pH. The presence of LMP80 was determined by the ligand blotting assay.
LEU-M3 we observed sCD14 bound to the LMP80/ lipid A complex. When using the anti-CD14 mAb clone MEM-18, which recognizes the LPS-binding epitope in the CD14 molecule [38], no sCD14 was detectable (Fig. 3).
Fig. 5. A¤nity chromatography on WGA for LMP80 puri¢cation. The LMP80-containing fractions of the IEF were pooled and applied to a WGA column. After washing, the column was eluted with 500 mM N-acetylglucosamine and every second of the obtained fractions was analyzed for protein concentration (upper panel), for purity by silver staining (A), and for LMP80 presence by the ligand blotting assay (B). LMP80 was found in the passthrough fractions, whereas the contaminants bound to the column and were eluted by GlcNAc. (A = applied sample, M = marker).
Fig. 6. Final LMP80 puri¢cation by preparative SDS-PAGE. The LMP80-containing fractions of the WGA chromatography were pooled and applied to preparative SDS-PAGE in a PrepCell. Every sixth fraction was analyzed for purity on a silverstained gel and for LMP80 presence by the ligand blotting assay. (A = applied sample, M = marker, LMP80-positive fractions from WGA chromatography).
3.4. Puri¢cation of LMP80 Because of the easy access to large amounts of erythrocytes, LMP80 was puri¢ed from these cells. Erythrocyte membrane proteins were extracted with the non-ionic detergent L-octylglycoside. The extracted proteins were further separated by preparative isoelectric focusing in a pH gradient from pH 3 to 10 as described in Section 2. After preparative isoelectric focusing, the presence of LMP80 in the fractions was determined by its lipid A-binding activity in a ligand blotting assay. Fig. 4 shows that LMP80 mainly accumulated in fractions 4^6, indicating an isoelectric point of about 5. Most of the other proteins accumulated in the basic fractions (fractions 15^20). In previous studies, we found that LMP80 is a glycoprotein (unpublished observation). Therefore, we considered further enrichment of LMP80 by af¢nity chromatography using the lectin WGA. WGA chromatography of the pooled LMP80-positive fractions of the IEF resulted in removal of most of contaminating proteins (see Figs. 5 and 5A). Surprisingly, LMP80 did not bind to WGA and was found only in the passthrough fractions and the ¢rst washing fractions (Fig. 5B, fractions 9^15). After SDS-PAGE and silver staining we found a band of about 80 kDa in the LMP80-containing fractions, but some other proteins of lower molecular mass
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Fig. 7. Expression of CD55 and LMP80 on CHO cells. A CD55-cDNA was constructed and expressed in CHO cells. Transfection e¤ciency was analyzed by FACS analysis (A) and by immunoblotting with anti-CD55 mAb (B). The presence of LMP80 was determined by the ligand blotting assay (C). (Lane 1: Erythrocyte extract ; lane 2: CHO-pCEP4 cells; lane 3; CHO-CD55 cells).
were also present. Most of these contaminating proteins, especially proteins of molecular masses in the range of LMP80, bound to WGA and were detected in the eluate fraction (Fig. 5A, fraction 27). The low-molecular-mass proteins in the LMP80containing fractions were then eliminated in a ¢nal puri¢cation step by preparative SDS-PAGE. Proteins were separated by their molecular mass and the puri¢ed LMP80 was found in the fractions 55^ 61 (Fig. 6). These pooled fractions showed only one clear protein band in a silver-stained gel (data not shown). 3.5. Amino acid sequencing of puri¢ed LMP80 Internal amino acid sequencing of the puri¢ed LMP80 was performed after digestion of LMP80 with endoproteinase Lys-C. It resulted in the following partial sequence: Q-P-Y-I-T-Q-N-Y-F-P. On sequence comparison to the SwissProt database it was found to have 100% identity to human CD55 (accession number P49457, positions 70^79).
3.6. Cloning of CD55 and expression in CHO-cells CHO-CD55 cells were prepared in order to serve for an additional con¢rmation of the identity of LMP80 and CD55. We constructed a cDNA encoding the sequence for CD55. An expression vector (pCEP4) containing this sequence was transfected into Chinese hamster ovary ¢broblasts (CHO-CD55). A control cell line was transfected with the cloning vector alone (CHO-pCEP4). After selection of positive transformants, cells were assayed for their CD55 expression by immuno£uorescence, using an anti-CD55 mAb (Fig. 7A). To con¢rm our previous results that CD55 is identical with LMP80, we analyzed the CD55-transfected CHO cells for the presence of LMP80 in the ligand blotting assay. As shown in Fig. 7B,C, the CD55-negative CHO cells became positive for both CD55 (Fig. 7B, lane 3) and LMP80 (Fig. 7C, lane 3), after transfection with CD55-cDNA. In contrast, the control transfectants with the empty vector remained CD55- and LMP80-negative (Fig. 7B,C, lane 2).
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4. Discussion A wide variety of mammalian cells can be activated by LPS. A prerequisite for this activation is the interaction between LPS and speci¢c cell surface receptors which are able to transmit the activation signal into the cell. The main target structure on monocytes/macrophages and neutrophils for LPS is the 55-kDa membrane protein mCD14 [7,8]. Nevertheless, the discovery of mCD14 as the major receptor for LPS and other bacterial structures raises new questions as to the mechanism of signal transduction after binding to mCD14. The lack of a transmembrane domain in the mCD14 molecule indicates that it may be incapable of transducing a signal on its own [39,40], suggesting the presence of other LPSresponsive elements. Furthermore, anti-CD14 antibodies inhibit LPS-induced activation of monocytes only when low amounts of LPS are applied [11,12]. Higher doses of LPS may omit the `high a¤nity receptor' mCD14 and bind directly to other target structures on the cell surface. Finally, a receptor recognizing sCD14/LPS complexes, which are capable of stimulating certain mCD14-negative cells, has been postulated to be present on these cells [13,14]. Several molecules are under discussion to serve as additional LPS receptors. The L-integrins were shown to bind LPS and to mediate the activation of NF-UB upon LPS stimulation [16^18]. Very recently the human Toll-like receptor-2 was identi¢ed as an LPS-responsive element downstream of CD14 [19]. In previous studies, we examined membrane proteins for LPS binding to select candidates for such hypothetical molecules. We found a membrane protein of about 80 kDa, which is able to bind LPS in a ligand blotting assay (LMP80) [20], provided sCD14 and LBP are present. Binding of LPS to LMP80 was also shown in a more physiological manner, by immuno-coprecipitation of LMP80 with anti-LPS antibodies after incubation of cell membranes (erythrocytes or monocytes) with LPS [21]. In the present report, we provide additional data about the expression of LMP80 and the formation of complexes consisting of LMP80/lipid A and sCD14 after lipid A binding. Furthermore, we describe the puri¢cation of LMP80 and its identi¢cation as the complement regulatory protein CD55 or DAF.
We found LMP80 expressed on monocytes as well as on all investigated monocytic cell lines. However, two cell lines, HL-60 and THP-1, exhibited a very weak expression. Furthermore, also other peripheral blood cells (granulocytes, T-lymphocytes, and erythrocytes) and vascular cells (smooth muscle cells and endothelial cells) were found to be LMP80-positive. With regard to a possible functional role of LMP80 it should be noted that the myeloid cell lines MonoMac-6, U937, HL-60 and THP-1 were able to respond to LPS [41^44]. Monocyte and granulocyte activation by LPS has been described in several publications [45], whereas the proliferation of T-lymphocytes in response to LPS was shown more recently [34]. Of special interest is the presence of LMP80 on vascular cells, since these cells respond to LPS only when present in complexes with the soluble CD14 molecule [13^15]. However, one cell line investigated so far lacks LMP80, the CHO ¢broblasts (Fig. 7), which are unable to respond to LPS [46]. When the ligand blotting assay for the detection of LMP80 was performed with various lipid A concentrations, we found a high sensitivity of binding of lipid A to LMP80 which is still detectable with minute amounts of lipid A (1 ng ml31 ) when serum is present (Fig. 2). When lipid A is present in higher concentrations ( s 10 Wg ml31 ) it can bind to LMP80 without the support of serum proteins. Binding of high LPS doses to human monocytes and their activation in response to this binding is also independent from the CD14 and LBP molecules [11,12]. This equal binding behavior leads us to the assumption that LMP80 is a good candidate for an accessory molecule beside or downstream of CD14. This suggestion is strongly supported by the ¢nding that also sCD14 is present in the complex consisting of lipid A and LMP80. The inability of the anti-CD14 mAb clone MEM-18 to detect sCD14 in the LMP80/ LPS/sCD14 complex indicates that the same epitope of CD14, which is responsible for the recognition of LPS by monocytes, is blocked by LPS molecules. In contrast, anti-CD14 mAbs, which recognize other epitopes in the CD14 molecule, were able to recognize sCD14 in the complex. The biochemical puri¢cation of LMP80 was achieved in a four-step procedure using erythrocyte membranes, which are abundantly available. This
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procedure includes IEF, WGA chromatography and preparative SDS-PAGE, which resulted in a single protein band in silver-stained gels. For the characterization of the puri¢ed protein, amino acid sequencing of an LMP80-derived peptide was performed giving ¢rst evidence of the identity of LMP80 and CD55. Immunochemical investigations using commercially available anti-CD55 mAbs con¢rmed the identity of CD55 with LMP80 (data not shown). In addition, we cloned the CD55-cDNA and expressed this molecule on CHO cells (Fig. 7). The ¢nding that only CD55-transfected CHO cells, in contrast to the vector transfectants, became positive for LMP80 after transfection with CD55-cDNA additionally demonstrated that LMP80 is identical to CD55. The identi¢cation of LMP80 as CD55 raises some new questions. CD55, like mCD14, is a GPI-linked molecule and therefore does not meet all requirements necessary for the supposed signal-transducing molecule downstream of CD14. Nevertheless, there are several reports dealing with the involvement of CD55 in signal transduction events [25^28,47]. In the case of human T-lymphocytes it was found that crosslinking of CD55 leads to proliferation, when cells are co-stimulated with phorbol esters [26]. After exposure to anti-CD55 mAbs, in human monocytes the glucose consumption and the phagocytosis of latex beads are increased [27]. It should be noticed, however, that no cytokine release was observed. Another report shows an enhanced calcium £ux and oxidative burst in human monocytes after crosslinking of CD55 by antibodies [48]. These results indicate that CD55 is a membrane molecule able to mediate activation of cells. However, the involvement of CD55 in the activation of in£ammatory cells by LPS remains to be investigated. CD55 transfected CHO cells now give us the basis for further investigations concerning this question.
Acknowledgments We want to thank Dr. R. Kroëncke for the FACS analysis, Dr. H. Brade (Research Center Borstel, Borstel, Germany) for providing the lipid A preparations, and K. Klopfenstein and I. Goroncy for
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their excellent technical assistance. We also want to thank Dr. V. Horejsi (Institute of Molecular Genetics, Prague, Czech Republic) for providing the anti-CD14 mAb MEM-18. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 367, Projects C5 to A.J.U. and B2 to U.M.), by the Bundesministerium fuër Bildung, Wissenschaft, Forschung und Technologie (BMBF Grant 01KI9471 to A.J.U.) and the Fonds der Chemischen Industrie (H.-D.F.). References [1] Rietschel, E.T. and Brade, H. (1992) Bacterial endotoxins. Sci. Am. 267, 54^61. [2] Rietschel, E.T., Brade, H., Holst, O., Brade, L., Muller-Loennies, S., Mamat, U., Zahringer, U., Beckmann, F., Seydel, U., Brandenburg, K., Ulmer, A.J., Mattern, T., Heine, H., Schletter, J., Loppnow, H., Schonbeck, U., Flad, H.D., Hauschildt, S., Schade, U.F., Di, P.F., Kusumoto, S. and Schumann, R.R. (1996) Bacterial endotoxin : Chemical constitution, biological recognition, host response, and immunological detoxi¢cation. Curr. Top. Microbiol. Immunol. 216, 39^81. [3] Galanos, C., Luderitz, O., Rietschel, E.T., Westphal, O., Brade, H., Brade, L., Freudenberg, M., Schade, U., Imoto, M. and Yoshimura, H. (1985) Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities. Eur. J. Biochem. 148, 1^5. [4] Loppnow, H., Brade, L., Brade, H., Rietschel, E.T., Kusumoto, S., Shiba, T. and Flad, H.D. (1986) Induction of human interleukin 1 by bacterial and synthetic lipid A. Eur. J. Immunol. 16, 1263^1267. [5] Loppnow, H., Libby, P., Freudenberg, M., Krauss, J.H., Weckesser, J. and Mayer, H. (1990) Cytokine induction by lipopolysaccharide (LPS) corresponds to lethal toxicity and is inhibited by nontoxic Rhodobacter capsulatus LPS. Infect. Immun. 58, 3743^3750. [6] Morrison, D.C. and Ryan, J.L. (1987) Endotoxins and disease mechanisms. Annu. Rev. Med. 38, 417^32. [7] Couturier, C., Jahns, G., Kazatchkine, M.D. and Hae¡nerCavaillon, N. (1992) Membrane molecules which trigger the production of interleukin-1 and tumor necrosis factor-alpha by lipopolysaccharide-stimulated human monocytes. Eur. J. Immunol. 22, 1461^1466. [8] Dentener, M.A., Bazil, V., Von, A.E., Ceska, M. and Buurman, W.A. (1993) Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-alpha, IL-6 and IL-8 release by human monocytes and alveolar macrophages. J. Immunol. 150, 2885^2891. [9] Hailman, E., Lichenstein, H.S., Wurfel, M.M., Miller, D.S., Johnson, D.A., Kelley, M., Busse, L.A., Zukowski, M.M. and Wright, S.D. (1994) Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J. Exp. Med. 179, 269^277.
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[10] Schumann, R.R., Leong, S.R., Flaggs, G.W., Gray, P.W., Wright, S.D., Mathison, J.C., Tobias, P.S. and Ulevitch, R.J. (1990) Structure and function of lipopolysaccharide binding protein. Science 249, 1429^1431. [11] Cohen, L., Haziot, A., Shen, D.R., Lin, X.Y., Sia, C., Harper, R., Silver, J. and Goyert, S.M. (1995) CD14-independent responses to LPS require a serum factor that is absent from neonates. J. Immunol. 155, 5337^5342. [12] Beaty, C.D., Franklin, T.L., Uehara, Y. and Wilson, C.B. (1994) Lipopolysaccharide-induced cytokine production in human monocytes: role of tyrosine phosphorylation in transmembrane signal transduction. Eur. J. Immunol. 24, 1278^ 1284. [13] Frey, E.A., Miller, D.S., Jahr, T.G., Sundan, A., Bazil, V., Espevik, T., Finlay, B.B. and Wright, S.D. (1992) Soluble CD14 participates in the response of cells to lipopolysaccharide. J. Exp. Med. 176, 1665^1671. [14] Loppnow, H. (1994) LPS, recIL1 and smooth muscle cell-IL1 activate vascular cells by speci¢c mechanisms. Prog. Clin. Biol. Res. 388, 309^321. [15] Delude, R.L., Savedra, R.J., Zhao, H., Thieringer, R., Yamamoto, S., Fenton, M.J. and Golenbock, D.T. (1995) CD14 enhances cellular responses to endotoxin without imparting ligand-speci¢c recognition. Proc. Natl. Acad. Sci. USA 92, 9288^9292. [16] Ingalls, R.R. and Golenbock, D.T. (1995) CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide. J. Exp. Med. 181, 1473^1479. [17] Ingalls, R.R., Arnaout, M.A., Delude, R.L., Flaherty, S., Savedra, R.J. and Golenbock, D.T. (1998) The CD11/CD18 integrins: characterization of three novel LPS signaling receptors. Prog. Clin. Biol. Res. 397, 107^117. [18] Flaherty, S.F., Golenbock, D.T., Milham, F.H. and Ingalls, R.R. (1997) CD11/CD18 leukocyte integrins : new signaling receptors for bacterial endotoxin. J. Surg. Res. 73, 85^89. [19] Yang, R.B., Mark, M.R., Gray, A., Huang, A., Xie, M.H., Zhang, M., Goddard, A., Wood, W.I., Gurney, A.L. and Godowski, P.J. (1998) Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling. Nature 395, 284^288. [20] Schletter, J., Brade, H., Brade, L., Kruger, C., Loppnow, H., Kusumoto, S., Rietschel, E.T., Flad, H.D. and Ulmer, A.J. (1995) Binding of lipopolysaccharide (LPS) to an 80-kilodalton membrane protein of human cells is mediated by soluble CD14 and LPS-binding protein. Infect. Immun. 63, 2576^ 2580. [21] El-Samalouti, V.T., Schletter, J., Brade, H., Brade, L., Kusumoto, S., Rietschel, E.T., Flad, H.D. and Ulmer, A.J. (1997) Detection of lipopolysaccharide (LPS)-binding membrane proteins by immuno-coprecipitation with LPS and anti-LPS antibodies. Eur. J. Biochem. 250, 418^424. [22] Kinoshita, T., Medof, M.E., Silber, R. and Nussenzweig, V. (1985) Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria. J. Exp. Med. 162, 75^92. [23] Nicholson-Weller, A. and Wang, C.E. (1994) Structure and function of decay accelerating factor CD55. J. Lab. Clin. Med. 123, 485^491.
[24] Fujita, T., Inoue, T., Ogawa, K., Iida, K. and Tamura, N. (1987) The mechanism of action of decay-accelerating factor (DAF). DAF inhibits the assembly of C3 convertases by dissociating C2a and Bb. J. Exp. Med. 166, 1221^1228. [25] Tosello, A.C., Mary, F., Amiot, M., Bernard, A. and Mary, D. (1998) Activation of T cells via CD55: recruitment of early components of the CD3-TCR pathway is required for IL-2 secretion. J. In£amm. 48, 13^27. [26] Davis, L.S., Patel, S.S., Atkinson, J.P. and Lipsky, P.E. (1988) Decay-accelerating factor functions as a signal transducing molecule for human T cells. J. Immunol. 141, 2246^ 2252. [27] Shibuya, K., Abe, T. and Fujita, T. (1992) Decay-accelerating factor functions as a signal transducing molecule for human monocytes. J. Immunol. 149, 1758^1762. [28] Shenoy-Scaria, A.M., Kwong, J., Fujita, T., Olszowy, M.W., Shaw, A.S. and Lublin, D.M. (1992) Signal transduction through decay-accelerating factor. Interaction of glycosyl-phosphatidylinositol anchor and protein tyrosine kinases p56lck and p59fyn 1. J. Immunol. 149, 3535^ 3541. [29] Bazil, V., Horejsi, V., Baudys, M., Kristofova, H., Strominger, J.L., Kostka, W. and Hilgert, I. (1986) Biochemical characterization of a soluble form of the 53-kDa monocyte surface antigen. Eur. J. Immunol. 16, 1583^1589. [30] Kuhn, H.M., Brade, L., Appelmelk, B.J., Kusumoto, S., Rietschel, E.T. and Brade, H. (1992) Characterization of the epitope speci¢city of murine monoclonal antibodies directed against lipid A. Infect. Immun. 60, 2201^2210. [31] Galanos, C. and Luderitz, O. (1975) Electrodialysis of lipopolysaccharides and their conversion to uniform salt forms. Eur. J. Biochem. 54, 603^610. [32] Brade, L. and Brade, H. (1985) Characterization of two di¡erent antibody speci¢cities recognizing distinct antigenic determinants in free lipid A of Escherichia coli. Infect. Immun. 48, 776^781. [33] Boyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest. Suppl. 97, 77^89. [34] Mattern, T., Thanhauser, A., Reiling, N., Toellner, K.M., Duchrow, M., Kusumoto, S., Rietschel, E.T., Ernst, M., Brade, H. and Flad, H.D. (1994) Endotoxin and lipid A stimulate proliferation of human T cells in the presence of autologous monocytes. J. Immunol. 153, 2996^3004. [35] Ja¡e, E.A., Nachman, R.L., Becker, C.G. and Minick, C.R. (1973) Culture of human endothelial cells derived from umbilical veins. Identi¢cation by morphologic and immunologic criteria. J. Clin. Invest. 52, 2745^2756. [36] Ross, R. and Kariya, B. (1980) Morphogenesis of vascular smooth muscle in atherosclerosis and cell structure. In: Handbook of Physiology, The Cardiovascular System (Bohr, D.F., Somlyo, A.P. and Sparks, H.Y., Eds.), pp. 66^91. American Physiological Society, Bethesda, MD. [37] Thanhauser, A., Reiling, N., Bohle, A., Toellner, K.M., Duchrow, M., Scheel, D., Schluter, C., Ernst, M., Flad,
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H.D. and Ulmer, A.J. (1993) Pentoxifylline: a potent inhibitor of IL-2 and IFN-gamma biosynthesis and BCG-induced cytotoxicity. Immunology 80, 151^156. Juan, T.S., Hailman, E., Kelley, M.J., Busse, L.A., Davy, E., Empig, C.J., Narhi, L.O., Wright, S.D. and Lichenstein, H.S. (1995) Identi¢cation of a lipopolysaccharide binding domain in CD14 between amino acids 57 and 64. J. Biol. Chem. 270, 5219^5224. Lee, J.D., Kravchenko, V., Kirkland, T.N., Han, J., Mackman, N., Moriarty, A., Leturcq, D., Tobias, P.S. and Ulevitch, R.J. (1993) Glycosyl-phosphatidylinositol-anchored or integral membrane forms of CD14 mediate identical cellular responses to endotoxin. Proc. Natl. Acad. Sci. USA 90, 9930^ 9934. Ulevitch, R.J. and Tobias, P.S. (1994) Recognition of endotoxin by cells leading to transmembrane signaling. Curr. Opin. Immunol. 6, 125^130. Labeta, M.O., Durieux, J.J., Fernandez, N., Herrmann, R. and Ferrara, P. (1993) Release from a human monocyte-like cell line of two di¡erent soluble forms of the lipopolysaccharide receptor, CD14. Eur. J. Immunol. 23, 2144^2151. Daniel-Issakani, S., Spiegel, A.M. and Strulovici, B. (1989) Lipopolysaccharide response is linked to the GTP binding protein, Gi2, in the promonocytic cell line U937. J. Biol. Chem. 264, 20240^20247. Cordle, S.R., Donald, R., Read, M.A. and Hawiger, J. (1993) Lipopolysaccharide induces phosphorylation of MAD3 and
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activation of c-Rel and related NF-kappa B proteins in human monocytic THP-1 cells. J. Biol. Chem. 268, 11803^ 11810. Maehara, K., Kanayama, N., Halim, A., El, M.E., Oda, T., Fujita, M. and Terao, T. (1995) Down-regulation of interleukin-8 gene expression in HL60 cell line by human Kunitz-type trypsin inhibitor. Biochem. Biophys. Res. Commun. 206, 927^ 934. Watson, R.W., Redmond, H.P. and Bouchier-Hayes, D. (1994) Role of endotoxin in mononuclear phagocyte-mediated in£ammatory responses. J. Leukocyte Biol. 56, 95^103. Golenbock, D.T., Liu, Y., Millham, F.H., Freeman, M.W. and Zoeller, R.A. (1993) Surface expression of human CD14 in Chinese hamster ovary ¢broblasts imparts macrophage-like responsiveness to bacterial endotoxin. J. Biol. Chem. 268, 22055^22059. Parolini, I., Sargiacomo, M., Lisanti, M.P. and Peschle, C. (1996) Signal transduction and glycophosphatidylinositollinked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton-insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes. Blood 87, 3783^3794. Lund-Johansen, F., Olweus, J., Symington, F.W., Arli, A., Thompson, J.S., Vilella, R., Skubitz, K. and Horejsi, V. (1993) Activation of human monocytes and granulocytes by monoclonal antibodies to glycosylphosphatidylinositol-anchored antigens. Eur. J. Immunol. 23, 2782^2791.
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Identi¢cation of the 80-kDa LPS-binding protein (LMP80) as decay-accelerating factor (DAF, CD55) Volker T. El-Samalouti a , Jens Schletter b , Ines Chyla a , Arnd Lentschat a , Uwe Mamat a , Lore Brade a , Hans-Dieter Flad a , Artur J. Ulmer a; *, Lutz Hamann a
a
Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany b Cardiogene, Gentherapeutische Systeme AG, D-40699 Erkrath, Germany Received 15 October 1998; received in revised form 2 December 1998; accepted 3 December 1998
Abstract The activation of immunocompetent cells by lipopolysaccharide (LPS) during severe Gram-negative infections is responsible for the pathophysiological reactions, possibly resulting in the clinical picture of sepsis. Monocytes recognize LPS mainly through the LPS receptor CD14, however, other cellular binding structures have been assumed to exist. In previous studies, we have described an 80-kDa LPS-binding membrane protein (LMP80), which is present on human monocytes as well as endothelial cells. Here we demonstrate that LMP80 is widely distributed and that it formes complexes together with LPS and sCD14. Furthermore, we report on the biochemical purification of LMP80 and its identification as decay-accelerating factor, CD55, by amino acid sequencing and cloning techniques. Our results imply a new feature of CD55 as a molecule which interacts with LPS/sCD14 complexes. However, the involvement of CD55 in LPS-induced signaling remains to be elucidated. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : CD55 ; Decay-accelerating factor ; Endotoxin; Lipopolysaccharide ; Lipopolysaccharide-binding protein; LMP80
1. Introduction Lipopolysaccharide (LPS, endotoxin), the major component of the outer lea£et of Gram-negative bacteria, is a potent inducer of an in£ammatory response [1,2]. LPS consists of a polysaccharide region * Corresponding author. Tel.: +49 (4537) 188448; Fax: +49 (4537) 188435; E-mail: [email protected] Abbreviations: DAF, decay-accelerating factor; LBP, lipopolysaccharide-binding protein; LMP80, 80-kDa LPS-binding membrane protein; NHS, normal human serum; PVA, polyvinyl alcohol
and a lipid part, called lipid A, which carries all endotoxic properties of the molecule [3,4]. LPS can lead to stimulation of immunocompetent cells, in particular monocytes/macrophages, resulting in the release of immune mediators such as the interleukins (IL) IL-1, IL-6, IL-8, IL-12 and tumor necrosis factor K (TNFK) [5,6]. The activation of cells through LPS requires the interaction of LPS with speci¢c cell surface molecules, capable of initiating transmembrane signaling. It is undisputed that the main binding site of LPS on monocytes/macrophages and granulocytes is the 55-kDa, glycosylphosphatidylinositol (GPI)-anchored CD14 molecule [7,8]. LPS bind-
0928-8244 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 9 8 ) 0 0 1 4 5 - X
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ing to membrane-linked CD14 (mCD14) as well as to the soluble form of CD14 (sCD14) is mediated by LPS-binding protein (LBP), an acute phase reactant present in serum [9,10]. However, there are many indications suggesting the presence of an accessory molecule beside or downstream of mCD14 involved in LPS signaling. Because it lacks a transmembrane domain, mCD14 alone seems to be incapable of transmitting the signal through the membrane. Furthermore, monocyte activation with high LPS doses cannot be blocked by anti-CD14 antibodies [11,12]. Therefore, it is assumed that CD14 mediates the recognition of low LPS doses, whereas high doses of LPS interact directly with another molecule. Additionally, cells of non-hematopoietic origin, such as endothelial cells or ¢broblasts, are responsive to LPS/sCD14 complexes [13^15], which indicates the existence of at least one target structure for the speci¢c recognition of sCD14/LPS complexes on these cells. Several molecules have been described that bind LPS, but an involvement in signal transduction events was only shown for the L-integrins [16^18] and recently for the human Toll-like receptor-2 [19]. In the past, we described a possible candidate for an additional LPS receptor connected to CD14, i.e. an 80-kDa LPS-binding membrane protein (LMP80), which binds LPS in a ligand blotting assay only in the presence of sCD14 and LBP [20]. We found LMP80 expressed on hematopoietic cells (monocytes, erythrocytes) as well as on non-hematopoietic cells (endothelial cells) [20,21]. Furthermore, we con¢rmed the interaction of LPS with LMP80 in its natural membranous environment by immuno-coprecipitation [21]. For this technique, cell membranes were incubated with LPS, solubilized by mild detergent treatment and the complexes of LPS and LMP80 were coprecipitated by the use of anti-LPS monoclonal antibodies (mAbs), indicating an association between the two molecules. The results presented in this paper provide proof that LMP80 is identical to CD55, the decay-accelerating factor (DAF). CD55 is a complement regulatory molecule, present on almost all cells that are in contact with the blood stream [22,23]. It protects the cells from autologous complement lysis by accelerating the decay of the C3 convertases of the classical and the alternative complement pathways [24].
Although CD55, like mCD14, is linked to the cell membrane by a GPI anchor in the cell membrane, it has repeatedly been shown to be involved in signal transduction on T-cells as well as on monocytes [25^ 28]. A functional involvement of CD55 in LPS induced signaling, however, remains to be investigated in further studies.
2. Materials and methods 2.1. Reagents and antibodies Unless otherwise indicated all ¢ne chemicals were purchased from Sigma (Deisenhofen, Germany), Serva (Heidelberg, Germany), Merck (Darmstadt, Germany), Bio-Rad (Munich, Germany), Gibco-BRL (Eggenstein, Germany), or Boehringer Mannheim (Mannheim, Germany). The anti-CD55 antibody IA10 was from Pharmingen (San Diego, CA, USA). Anti-CD14 antibodies were from Immunotech (IOM2), Becton Dickinson (Leu-M3) or Biometec (ROMO-1). MEM-18 [29] was kindly provided by Dr. V. Horejsi (Institute of Molecular Genetics, Prague, Czech Republic). Anti-lipid A mAb A6, which recognizes the phosphorylated carbohydrate backbone of lipid A independently of the acylation pattern, was used as hybridoma serum-free supernatant [30]. Peroxidase-conjugated or FITC-labeled goat antimouse immunoglobulin G was purchased from Dianova (Hamburg, Germany). IgG 2a and IgG 2b isotype control antibodies were purchased from Sigma. Normal human sera were obtained from healthy volunteers. LPS from Escherichia coli Re mutant F515 was prepared by the hot phenol-water procedure, puri¢ed by repeated ultracentrifugation, and converted into the sodium salt form by electrodialysis [31]. Lipid A from E. coli Re mutant F515 was prepared by treating LPS with acetate bu¡er [32]. All LPS and lipid A preparations were solubilized in pyrogen-free distilled water and stored in aliquots at 1 mg ml31 and 4³C. Oligonucleotides were synthesized by TIB Molbiol (Berlin, Germany) or MWG (Munich, Germany). Cloning vectors pCR II and pCEP4 were obtained from Invitrogen (Leek, The Netherlands).
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2.2. Cell separation Human blood cells from healthy donors were separated by density gradient centrifugation of heparinized blood with Ficoll-Paque [33] into two fractions, mononuclear cells and erythrocytes/ granulocytes. For erythrocyte preparation the pellet of the density gradient centrifugation was harvested without the granulocyte layer, washed three times with RPMI 1640 and resuspended in an equal volume of RPMI 1640. The cells were applied to a dextran cushion (3% dextran in 150 mM NaCl) and sedimented at room temperature for 18 min. The erythrocytes were collected and washed two times with PBS. For the isolation of granulocytes, the overlayer of the erythrocyte pellet was applied to a PVA cushion (1% PVA in 0.9% NaCl) and sedimented at room temperature for 20 min. The pellet was collected and erythrocytes were lysed by incubation in 5 ml distilled water. After 45 s 5 ml of 2UPBS was added. Monocytes were isolated by counter£ow elutriation of mononuclear cells as described [20], T-lymphocytes, endothelial cells and smooth muscle cells were prepared as described elsewhere [34^36]. 2.3. Cell culture The monocytic cell line Mono-Mac-6 was kindly provided by Dr. H.W.L. Ziegler-Heitbrock (Institute of Immunology, University of Munich, Munich, Germany). The monocytic cell lines THP-1 and HL-60 were from the American Tissue Culture Collection. All monocytic cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, penicillin (100 U ml31 ), and streptomycin (100 Wg ml31 ). CHO-K1 cells from the DSMZ (Braunschweig, Germany) were cultured in Ham's F12 medium supplemented with 10% fetal calf serum, penicillin (100 U ml31 ), and streptomycin (100 Wg ml31 ). Transfectants were cultured in the presence of 400 Wg ml31 hygromycin. All cultures were maintained in a humidi¢ed incubator at 37³C and 5% CO2 . 2.4. Cell membrane preparation Packed erythrocytes (10 ml) were resuspended in 260 ml hypotonic lysis bu¡er (10 mM Tris-HCl, pH
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7.4) and shaken for 15 min on ice. The lysate was centrifuged at 18 000Ug, 15 min, 4³C, the pellet was washed once with hypotonic lysis bu¡er and twice with distilled water. The membranes were pelleted by a ¢nal centrifugation step (50 000Ug, 35 min, 4³C) and stored at 320³C. Membranes of all other cells and human cell lines were prepared as described elsewhere [20]. CHO cells were harvested after washing monolayers with PBS by incubation with Cell Dissociation Solution (Sigma, Deisenhofen). 50U106 cells were resuspended in 1 ml ice cold lysis bu¡er (10 mM Tris-HCl, pH 7.4 with 1 mM phenylmethylsulfonyl £uoride (PMSF), 2 mM EDTA) and subsequently sonicated on ice in a Branson soni¢er 250 (10 strokes, intensity 1.1, duty cycle 80%). After removal of debris by centrifugation (400Ug, 15 min, 4³C), enriched cell membrane fractions were obtained by high speed centrifugation (36 000Ug, 30 min, 4³C), and aliquots were stored at 320³C. 2.5. Electrophoresis, immunoblotting and ligand blotting assay Probes were subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (LMP80 and CD14 detection) or under non-reducing conditions (CD55 detection) on a 10% gel, followed by blotting of the proteins onto nitrocellulose membranes using a semidry blotting apparatus for 2 h at 2.5 mA cm32 . Nitrocellulose membranes were blocked for 2 h at room temperature with PBS containing 5% non-fat dry milk (PBS/M). The ligand blotting assay for the detection of LMP80 was performed as described elsewhere with some modi¢cations [20]. Brie£y, blocked membranes were incubated for 1.5 h with 0.5 Wg ml31 lipid A in PBS/M-0.1% L-octylglycoside in the presence or absence of 3% NHS. After four washes in PBS containing 0.05% Tween 20 (PBS/T), bound lipid A was detected by anti-lipid A mAb A6. For the detection of CD55, blocked membranes were incubated with anti-CD55 mAb IA10 (1:5000) in PBS/M overnight at 4³C. On the next day, immunoblots were washed four times in PBS/T, incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse mAb in PBS/
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M, and developed with the ECL system (Amersham Buchler, Braunschweig, Germany). 2.6. Puri¢cation of LMP80 Erythrocyte membranes containing 40 mg protein were solubilized in 20 ml solubilization bu¡er (2.5% L-octylglycoside, 1 mM PMSF, 1 mM EDTA) for 30 min at 4³C. After centrifugation (100 000Ug, 30 min, 4³C) supernatants were supplemented to a ¢nal volume of 50 ml with 5% glycerol, 1 g L-octylglycoside, urea (to 2 M), PMSF (to 1 mM), ampholytes (40% Biolyte 3-10 and 40% Biolyte 4-6). Preparative isoelectric focusing (IEF) was performed at 4³C and 12 W in a Rotofer cell (Bio-Rad, Munich, Germany) for 5 h. The LMP80-containing fractions as detected by ligand blotting assay were pooled, washed with equilibration bu¡er (50 mM NaH2 PO4 , 500 mM NaCl, 0.1 mM CaCl2 , 0.1 mM MgCl2 , 1% L-octylglycoside, pH 7), and concentrated in a Centricon-50 (Amicon, Beverly, MA, USA). LMP80 was further enriched by a¤nity chromatography on a column of wheat germ agglutinin (WGA) coupled to agarose. The column was loaded with the concentrated fractions from IEF (2 ml h31 ), washed with equilibration bu¡er (10 ml at 4 ml h31 ), and eluted with elution bu¡er (equilibration bu¡er with 0.5 M WGA). 0.5ml fractions were collected, protein bands were detected by silver staining, and the presence of LMP80 was checked using the ligand blotting assay. LMP80containing fractions were then pooled, concentrated in a Centricon-50 (Amicon, Beverly, MA, USA), and further puri¢ed on a 9% preparative SDS-PAGE in a Model 491 Prep Cell (Bio-Rad, Munich, Germany) at 40 mA. 2.5-ml fractions were collected and the purity of LMP80 was estimated to be greater than 95% by determination on a silver-stained gel. 2.7. Amino acid sequencing Amino acid sequencing of LMP80 was performed by Eurogentec (Seraing, Belgium). 2.8. Generation of CD55 cDNA and cell transfection Monocyte mRNA was isolated from 2U106 cells using oligo-dT-coated magnetic beads (Dynal, Hamburg, Germany), according to the manufacturer's in-
structions, and cDNA was prepared by subsequent reverse transcription as described [37]. Ampli¢cation of CD55 cDNA was carried out using an automatic DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT, USA), and the oligonucleotide pair (5P ATA TGG ATC CAC CAT GAC CGT CGC GCG GCC GAG C+5P ATA TTC TAG ACT AAG AAA CTA GGA ACA GTC TGT ATA CTT) used was designed to introduce a BamHI site, a Kozak sequence, and an XbaI site into the product. The PCR conditions were: 20 cycles, annealing temperature 60³C. The PCR product was cloned in E. coli DH5K employing the TA vector pCR II (Invitrogen). Positive clones were sequenced with the USB Sequenase 2.0 Kit (Amersham Buchler, Braunschweig, Germany) and subcloned into the KpnI and XbaI sites of the vector pCEP4 (pCEP4-CD55). Control sequencing of the ¢nal construct was carried out at MWG (Ebersbach, Germany). CHO cells were transfected with pCEP4 (CHOpCEP4) or pCEP4-CD55 (CHO-CD55) by using Fugene1 (Boehringer Mannheim) as described in the manufacturer's protocol, and selection of positive transfectants was performed after 48 h with hygromycin (400 Wg ml31 ). CD55-positive cells were enriched ( s 80%) by cell sorting with a FACSstar (Becton Dickinson) using anti-CD55 mAbs.
3. Results 3.1. Expression of LMP80 in di¡erent cell types In previous studies we have found that LMP80 is expressed on monocytic cells as well as on endothelial cells and erythrocytes. Furthermore, we checked several other myeloid and non-myeloid cells and cell lines for their LMP80 expression. We analyzed cell membrane preparations containing 30 Wg of total protein by the ligand blotting assay (Fig. 1). We found that LMP80 is present in all tested cells but there are striking di¡erences in the protein quantity (Fig. 1). The amount of LMP80 was similar in monocytes, T-lymphocytes, erythrocytes, granulocytes, endothelial cells, and in the cell lines MonoMac-6 and U937. Smooth muscle cells and the promyeloid cell lines THP-1 and HL-60 showed only weak LMP80 expression. It should be noted that
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Fig. 1. Expression of LMP80. Cell membrane preparations containing 30 Wg of total protein were separated by SDS-PAGE and blotted. Lanes were split, incubated with lipid A (0.5 Wg ml31 ) in the presence (+) or in the absence (3) of normal human serum (NHS) and bound lipid A was detected by anti-lipid A mAb. LMP80 was principally expressed on all investigated cells types, but THP-1 and HL-60 cells contained only minute amounts of LPM80.
the ligand blotting assay was performed under reducing conditions and, therefore, binding of lipid A to CD14 on CD14-positive cells is not observed. 3.2. Dose-dependent binding of lipid A to LMP80 In further studies we analyzed the in£uence of lipid A concentration on the binding of lipid A to LMP80. We found that minimal concentrations (1 ng ml31 ) of lipid A are su¤cient to bind to LMP80 in a serum-dependent manner (Fig. 2). When high concentrations of lipid A were used, serum was not required for the mediation of the binding of lipid A to LMP80.
Fig. 2. Dose-dependent binding of lipid A to LMP80. MonoMac-6 membrane preparations containing 30 Wg of total protein were separated by SDS-PAGE and blotted. Lanes were split and incubated with various lipid A concentrations in the presence (+) or in the absence (3) of NHS. Bound lipid A was detected by anti-lipid A mAb. Binding of lipid A to LMP80 was detectable even in very low lipid A concentrations (1 ng ml31 ). High LPS doses ( s 10 Wg ml31 ) were capable of binding to LPM80 independently of sCD14.
3.3. Presence of sCD14 in the lipid A/LMP80 complex sCD14 is required to mediate together with LBP the binding of low amounts of lipid A to LMP80 [20]. We therefore analyzed whether sCD14 is physically present in the LMP80/lipid A complex. We incubated the electrophoretically separated and blotted proteins with lipid A in the presence or absence of serum. Instead of lipid A detection, we incubated those membranes with di¡erent anti-CD14 antibodies. With the anti-CD14 mAbs ROMO-1, IOM2, and
Fig. 3. Detection of sCD14 in the LMP80/lipid A complex. Mono-Mac-6 membrane preparations containing 30 Wg of total protein were separated by SDS-PAGE and blotted. Lanes were split and incubated with various lipid A concentrations in the presence (+) or in the absence (3) of NHS. Bound sCD14 was detected by anti-CD14 mAbs. The presence of sCD14 complexed to LMP80/lipid A was detected with the mAbs IOM2, Leu-M3 and Romo-1, whereas the clone MEM-18 failed in sCD14 detection.
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Fig. 4. Preparative IEF for LMP80 enrichment. Erythrocyte membranes were solubilized with L-octylglycoside and separated by isoelectric focusing in a Rotofor cell. The obtained fractions were analyzed for their protein concentration and their pH. The presence of LMP80 was determined by the ligand blotting assay.
LEU-M3 we observed sCD14 bound to the LMP80/ lipid A complex. When using the anti-CD14 mAb clone MEM-18, which recognizes the LPS-binding epitope in the CD14 molecule [38], no sCD14 was detectable (Fig. 3).
Fig. 5. A¤nity chromatography on WGA for LMP80 puri¢cation. The LMP80-containing fractions of the IEF were pooled and applied to a WGA column. After washing, the column was eluted with 500 mM N-acetylglucosamine and every second of the obtained fractions was analyzed for protein concentration (upper panel), for purity by silver staining (A), and for LMP80 presence by the ligand blotting assay (B). LMP80 was found in the passthrough fractions, whereas the contaminants bound to the column and were eluted by GlcNAc. (A = applied sample, M = marker).
Fig. 6. Final LMP80 puri¢cation by preparative SDS-PAGE. The LMP80-containing fractions of the WGA chromatography were pooled and applied to preparative SDS-PAGE in a PrepCell. Every sixth fraction was analyzed for purity on a silverstained gel and for LMP80 presence by the ligand blotting assay. (A = applied sample, M = marker, LMP80-positive fractions from WGA chromatography).
3.4. Puri¢cation of LMP80 Because of the easy access to large amounts of erythrocytes, LMP80 was puri¢ed from these cells. Erythrocyte membrane proteins were extracted with the non-ionic detergent L-octylglycoside. The extracted proteins were further separated by preparative isoelectric focusing in a pH gradient from pH 3 to 10 as described in Section 2. After preparative isoelectric focusing, the presence of LMP80 in the fractions was determined by its lipid A-binding activity in a ligand blotting assay. Fig. 4 shows that LMP80 mainly accumulated in fractions 4^6, indicating an isoelectric point of about 5. Most of the other proteins accumulated in the basic fractions (fractions 15^20). In previous studies, we found that LMP80 is a glycoprotein (unpublished observation). Therefore, we considered further enrichment of LMP80 by af¢nity chromatography using the lectin WGA. WGA chromatography of the pooled LMP80-positive fractions of the IEF resulted in removal of most of contaminating proteins (see Figs. 5 and 5A). Surprisingly, LMP80 did not bind to WGA and was found only in the passthrough fractions and the ¢rst washing fractions (Fig. 5B, fractions 9^15). After SDS-PAGE and silver staining we found a band of about 80 kDa in the LMP80-containing fractions, but some other proteins of lower molecular mass
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Fig. 7. Expression of CD55 and LMP80 on CHO cells. A CD55-cDNA was constructed and expressed in CHO cells. Transfection e¤ciency was analyzed by FACS analysis (A) and by immunoblotting with anti-CD55 mAb (B). The presence of LMP80 was determined by the ligand blotting assay (C). (Lane 1: Erythrocyte extract ; lane 2: CHO-pCEP4 cells; lane 3; CHO-CD55 cells).
were also present. Most of these contaminating proteins, especially proteins of molecular masses in the range of LMP80, bound to WGA and were detected in the eluate fraction (Fig. 5A, fraction 27). The low-molecular-mass proteins in the LMP80containing fractions were then eliminated in a ¢nal puri¢cation step by preparative SDS-PAGE. Proteins were separated by their molecular mass and the puri¢ed LMP80 was found in the fractions 55^ 61 (Fig. 6). These pooled fractions showed only one clear protein band in a silver-stained gel (data not shown). 3.5. Amino acid sequencing of puri¢ed LMP80 Internal amino acid sequencing of the puri¢ed LMP80 was performed after digestion of LMP80 with endoproteinase Lys-C. It resulted in the following partial sequence: Q-P-Y-I-T-Q-N-Y-F-P. On sequence comparison to the SwissProt database it was found to have 100% identity to human CD55 (accession number P49457, positions 70^79).
3.6. Cloning of CD55 and expression in CHO-cells CHO-CD55 cells were prepared in order to serve for an additional con¢rmation of the identity of LMP80 and CD55. We constructed a cDNA encoding the sequence for CD55. An expression vector (pCEP4) containing this sequence was transfected into Chinese hamster ovary ¢broblasts (CHO-CD55). A control cell line was transfected with the cloning vector alone (CHO-pCEP4). After selection of positive transformants, cells were assayed for their CD55 expression by immuno£uorescence, using an anti-CD55 mAb (Fig. 7A). To con¢rm our previous results that CD55 is identical with LMP80, we analyzed the CD55-transfected CHO cells for the presence of LMP80 in the ligand blotting assay. As shown in Fig. 7B,C, the CD55-negative CHO cells became positive for both CD55 (Fig. 7B, lane 3) and LMP80 (Fig. 7C, lane 3), after transfection with CD55-cDNA. In contrast, the control transfectants with the empty vector remained CD55- and LMP80-negative (Fig. 7B,C, lane 2).
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4. Discussion A wide variety of mammalian cells can be activated by LPS. A prerequisite for this activation is the interaction between LPS and speci¢c cell surface receptors which are able to transmit the activation signal into the cell. The main target structure on monocytes/macrophages and neutrophils for LPS is the 55-kDa membrane protein mCD14 [7,8]. Nevertheless, the discovery of mCD14 as the major receptor for LPS and other bacterial structures raises new questions as to the mechanism of signal transduction after binding to mCD14. The lack of a transmembrane domain in the mCD14 molecule indicates that it may be incapable of transducing a signal on its own [39,40], suggesting the presence of other LPSresponsive elements. Furthermore, anti-CD14 antibodies inhibit LPS-induced activation of monocytes only when low amounts of LPS are applied [11,12]. Higher doses of LPS may omit the `high a¤nity receptor' mCD14 and bind directly to other target structures on the cell surface. Finally, a receptor recognizing sCD14/LPS complexes, which are capable of stimulating certain mCD14-negative cells, has been postulated to be present on these cells [13,14]. Several molecules are under discussion to serve as additional LPS receptors. The L-integrins were shown to bind LPS and to mediate the activation of NF-UB upon LPS stimulation [16^18]. Very recently the human Toll-like receptor-2 was identi¢ed as an LPS-responsive element downstream of CD14 [19]. In previous studies, we examined membrane proteins for LPS binding to select candidates for such hypothetical molecules. We found a membrane protein of about 80 kDa, which is able to bind LPS in a ligand blotting assay (LMP80) [20], provided sCD14 and LBP are present. Binding of LPS to LMP80 was also shown in a more physiological manner, by immuno-coprecipitation of LMP80 with anti-LPS antibodies after incubation of cell membranes (erythrocytes or monocytes) with LPS [21]. In the present report, we provide additional data about the expression of LMP80 and the formation of complexes consisting of LMP80/lipid A and sCD14 after lipid A binding. Furthermore, we describe the puri¢cation of LMP80 and its identi¢cation as the complement regulatory protein CD55 or DAF.
We found LMP80 expressed on monocytes as well as on all investigated monocytic cell lines. However, two cell lines, HL-60 and THP-1, exhibited a very weak expression. Furthermore, also other peripheral blood cells (granulocytes, T-lymphocytes, and erythrocytes) and vascular cells (smooth muscle cells and endothelial cells) were found to be LMP80-positive. With regard to a possible functional role of LMP80 it should be noted that the myeloid cell lines MonoMac-6, U937, HL-60 and THP-1 were able to respond to LPS [41^44]. Monocyte and granulocyte activation by LPS has been described in several publications [45], whereas the proliferation of T-lymphocytes in response to LPS was shown more recently [34]. Of special interest is the presence of LMP80 on vascular cells, since these cells respond to LPS only when present in complexes with the soluble CD14 molecule [13^15]. However, one cell line investigated so far lacks LMP80, the CHO ¢broblasts (Fig. 7), which are unable to respond to LPS [46]. When the ligand blotting assay for the detection of LMP80 was performed with various lipid A concentrations, we found a high sensitivity of binding of lipid A to LMP80 which is still detectable with minute amounts of lipid A (1 ng ml31 ) when serum is present (Fig. 2). When lipid A is present in higher concentrations ( s 10 Wg ml31 ) it can bind to LMP80 without the support of serum proteins. Binding of high LPS doses to human monocytes and their activation in response to this binding is also independent from the CD14 and LBP molecules [11,12]. This equal binding behavior leads us to the assumption that LMP80 is a good candidate for an accessory molecule beside or downstream of CD14. This suggestion is strongly supported by the ¢nding that also sCD14 is present in the complex consisting of lipid A and LMP80. The inability of the anti-CD14 mAb clone MEM-18 to detect sCD14 in the LMP80/ LPS/sCD14 complex indicates that the same epitope of CD14, which is responsible for the recognition of LPS by monocytes, is blocked by LPS molecules. In contrast, anti-CD14 mAbs, which recognize other epitopes in the CD14 molecule, were able to recognize sCD14 in the complex. The biochemical puri¢cation of LMP80 was achieved in a four-step procedure using erythrocyte membranes, which are abundantly available. This
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procedure includes IEF, WGA chromatography and preparative SDS-PAGE, which resulted in a single protein band in silver-stained gels. For the characterization of the puri¢ed protein, amino acid sequencing of an LMP80-derived peptide was performed giving ¢rst evidence of the identity of LMP80 and CD55. Immunochemical investigations using commercially available anti-CD55 mAbs con¢rmed the identity of CD55 with LMP80 (data not shown). In addition, we cloned the CD55-cDNA and expressed this molecule on CHO cells (Fig. 7). The ¢nding that only CD55-transfected CHO cells, in contrast to the vector transfectants, became positive for LMP80 after transfection with CD55-cDNA additionally demonstrated that LMP80 is identical to CD55. The identi¢cation of LMP80 as CD55 raises some new questions. CD55, like mCD14, is a GPI-linked molecule and therefore does not meet all requirements necessary for the supposed signal-transducing molecule downstream of CD14. Nevertheless, there are several reports dealing with the involvement of CD55 in signal transduction events [25^28,47]. In the case of human T-lymphocytes it was found that crosslinking of CD55 leads to proliferation, when cells are co-stimulated with phorbol esters [26]. After exposure to anti-CD55 mAbs, in human monocytes the glucose consumption and the phagocytosis of latex beads are increased [27]. It should be noticed, however, that no cytokine release was observed. Another report shows an enhanced calcium £ux and oxidative burst in human monocytes after crosslinking of CD55 by antibodies [48]. These results indicate that CD55 is a membrane molecule able to mediate activation of cells. However, the involvement of CD55 in the activation of in£ammatory cells by LPS remains to be investigated. CD55 transfected CHO cells now give us the basis for further investigations concerning this question.
Acknowledgments We want to thank Dr. R. Kroëncke for the FACS analysis, Dr. H. Brade (Research Center Borstel, Borstel, Germany) for providing the lipid A preparations, and K. Klopfenstein and I. Goroncy for
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their excellent technical assistance. We also want to thank Dr. V. Horejsi (Institute of Molecular Genetics, Prague, Czech Republic) for providing the anti-CD14 mAb MEM-18. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 367, Projects C5 to A.J.U. and B2 to U.M.), by the Bundesministerium fuër Bildung, Wissenschaft, Forschung und Technologie (BMBF Grant 01KI9471 to A.J.U.) and the Fonds der Chemischen Industrie (H.-D.F.). References [1] Rietschel, E.T. and Brade, H. (1992) Bacterial endotoxins. Sci. Am. 267, 54^61. [2] Rietschel, E.T., Brade, H., Holst, O., Brade, L., Muller-Loennies, S., Mamat, U., Zahringer, U., Beckmann, F., Seydel, U., Brandenburg, K., Ulmer, A.J., Mattern, T., Heine, H., Schletter, J., Loppnow, H., Schonbeck, U., Flad, H.D., Hauschildt, S., Schade, U.F., Di, P.F., Kusumoto, S. and Schumann, R.R. (1996) Bacterial endotoxin : Chemical constitution, biological recognition, host response, and immunological detoxi¢cation. Curr. Top. Microbiol. Immunol. 216, 39^81. [3] Galanos, C., Luderitz, O., Rietschel, E.T., Westphal, O., Brade, H., Brade, L., Freudenberg, M., Schade, U., Imoto, M. and Yoshimura, H. (1985) Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities. Eur. J. Biochem. 148, 1^5. [4] Loppnow, H., Brade, L., Brade, H., Rietschel, E.T., Kusumoto, S., Shiba, T. and Flad, H.D. (1986) Induction of human interleukin 1 by bacterial and synthetic lipid A. Eur. J. Immunol. 16, 1263^1267. [5] Loppnow, H., Libby, P., Freudenberg, M., Krauss, J.H., Weckesser, J. and Mayer, H. (1990) Cytokine induction by lipopolysaccharide (LPS) corresponds to lethal toxicity and is inhibited by nontoxic Rhodobacter capsulatus LPS. Infect. Immun. 58, 3743^3750. [6] Morrison, D.C. and Ryan, J.L. (1987) Endotoxins and disease mechanisms. Annu. Rev. Med. 38, 417^32. [7] Couturier, C., Jahns, G., Kazatchkine, M.D. and Hae¡nerCavaillon, N. (1992) Membrane molecules which trigger the production of interleukin-1 and tumor necrosis factor-alpha by lipopolysaccharide-stimulated human monocytes. Eur. J. Immunol. 22, 1461^1466. [8] Dentener, M.A., Bazil, V., Von, A.E., Ceska, M. and Buurman, W.A. (1993) Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-alpha, IL-6 and IL-8 release by human monocytes and alveolar macrophages. J. Immunol. 150, 2885^2891. [9] Hailman, E., Lichenstein, H.S., Wurfel, M.M., Miller, D.S., Johnson, D.A., Kelley, M., Busse, L.A., Zukowski, M.M. and Wright, S.D. (1994) Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J. Exp. Med. 179, 269^277.
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[38]
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