Interactions of bacterial lipopolysaccharide and peptidoglycan with a 70 kDa and an 80 kDa protein on the cell surface of CD14+ and CD14− cells

Interactions of Bacterial Lipopolysaccharide and Peptidoglycan with a 70 kDa and an 80 kDa Protein on the Cell Surface of CD14ⴙ and CD14ⴚ Cells Kathy Triantafilou, Martha Triantafilou, and Russell L. Dedrick ABSTRACT: Bacterial cell wall components, lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) are known to stimulate cells of the immune, inflammatory and vascular systems contributing to septic shock. CD14 has been identified as the main LPS receptor, a process that is accelerated by the serum protein LPSbinding protein (LBP). CD14 has also been found to bind LTA and PGN from the cell wall of gram positive bacteria. Recently, toll-like receptor proteins TLR-2 and TLR-4 have been shown to be required for LPS and LTA-induced intracellular signalling. Although CD14 functions as either a glycosylphosphatidylinositol (GPI)anchored molecule that does not transverse the cell membrane or as a soluble serum protein, the mechanisms by which the CD14-LPS/LTA complex interacts with the TLRs remains to be elucidated. We have looked directly for cell surface protein(s) that bind LPS or LTA in a

ABBREVIATIONS CHO Chinese hamster ovary LAP LPS/LTA associated protein LBP LPS binding protein LPS lipopolysaccharide

INTRODUCTION The recognition of microbial pathogens is a complex and multistep process involving serum proteins and cells of the innate immune system. Gram-negative and -positive

From the Department of Biological Sciences (K.T., M.T.), University of Essex, Central Campus, Colchester, United Kingdom; and Molecular Immunology (R.L.D.), XOMA (US) LLC, Berkeley, CA, USA. Address reprint requests to: Kathy Triantafilou, Ph.D., Department of Biological Sciences, University of Essex, Central Campus, Wivenhoe Park, Colchester, CO4 3SQ, United Kingdom; Tel: ⫹44 (1206) 873787; Fax: ⫹44 (1206) 872592; E-mail: [email protected]. Received July 27, 2000; revised September 25, 2000; accepted October 2, 2000. Human Immunology 62, 50 – 63 (2001) © American Society for Histocompatibility and Immunogenetics, 2001 Published by Elsevier Science Inc.

CD14-dependent manner. Using biochemical approaches we have identified two proteins of molecular weight 70 kDa (LAP-1) and 80 kDa (LAP-2) that can be precipitated from both CD14⫹ and CD14⫺ cells with LPS- or LTAspecific antibodies. Binding of LPS and LTA to LAP-1 and -2 required serum. While soluble CD14 (sCD14) was sufficient to allow precipitation of these two proteins from CD14⫺ cells, serum could not be replaced by purified sCD14 and/or LBP when mCD14-expressing cells were used. Human Immunology 62, 50 – 63 (2001). © American Society for Histocompatibility and Immunogenetics, 2001. Published by Elsevier Science Inc. KEYWORDS: human; endothelial cells; endotoxin shock; infectious immunity-bacteria; lipopolysaccharide; biochemistry

LTA mCD14 PGN sCD14

lipoteichoic acid membrane CD14 peptidoglycan soluble CD14

bacteria represent a major group of pathogens causing human disease. Their recognition by the innate immune system leads to activation of monocytes and macrophages, and under certain circumstances can lead to the complex and life-threatening clinical syndrome of septic shock. The molecular mechanisms involved in innate recognition of bacteria still remains obscure. CD14, a cell surface molecule found on cells of myeloid lineage, anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, has been implicated in mediating inflammatory responses to many pathogen-derived ligands, including gram-positive bac0198-8859/01/$–see front matter PII S0198-8859(00)00222-6

Association of LPS/LTA with LAP Proteins

terial peptidoglycan (PGN) and lipoteichoic acid (LTA) [1, 2], which is the major component of the cell wall of gram-positive bacteria. In the case of gram-negative bacteria, a glycolipid, known as endotoxin or lipopolysaccharide (LPS), is a component of the outer membrane of all gram-negative bacteria was discovered to be the principal constituent that is recognized by the innate immune system. It was established that LPS-activated myeloid lineage cells by an unanticipated mechanism involving CD14 [3] and a serum protein, lipopolysaccharide binding protein (LBP) [4]. LBP is a 60 kDa serum glycoprotein that binds LPS via a lipid A, [5] and transfers LPS-LBP complexes to CD14 [3]. Because CD14 is a GPI anchored protein molecule and does not traverse the cell membrane, the mechanism by which it mediates the signal to the cytoplasm has remained a longstanding question in the field. It has been suggested that CD14 is a multimeric receptor [6] and plays a critical role in binding LPS, but at least one other additional transmembrane molecule is involved that represents the signaling unit of this multimeric receptor. Recent data provide support for this hypothesis by showing that members of the toll-like receptors (TLR) are involved in CD14-dependent LPS responses [7–14]. Another activation pathway exists that involves cells that lack membrane bound CD14 (mCD14) [15, 16]. Complexes of soluble CD14 (sCD14) and LPS bind to a yet unknown receptor and activate cells of nonmyeloid lineage [15, 17–20]. It has been suggested that the signalling receptor involved in mCD14-LPS and sCD14LPS complexes is possibly one and the same. In this study we sought to gain better insights into LPS/PGNCD14 binding and to determine which molecules at the cell surface may also be involved in LPS/PGN binding and signal transduction. Chinese Hamster Ovary (CHO) cells transfected with CD14 were used for these experiments, as well as endothelial cells, which are known not to express CD14. CHO cells are generally unresponsive to LPS, as measured by the activation of NF-␬B, and this response can only be potentiated by the transfection of mCD14 into CHO cells. CHO cells transfected with different N-terminal deletions of CD14 were also used to map CD14 epitopes involved in LPS/PGN interactions. Many experimental procedures have been used in order to detect LPS/PGN receptors on target cells. Including photochemical crosslinking of ASD-LPS [21–23], LPS-coated magnetic beads [24], or separation of LPS binding molecules by SDS-PAGE and transfer on nitrocellulose followed by incubation with LPS [25, 26]. In order to obtain a more physiologic detection system for LPS/PGN binding receptors, we developed an immunoprecipitation assay using LPS/lipoteichoic acid (LTA), with concentrations similar to those found in patients

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with sepsis and anti-LPS or LTA antibodies. LPS or LTA were used as an affinity surface allowing it to bind to the solubilized receptors in the presence of 5% human serum. The LPS/LTA receptor complexes were then immunoprecipitated by LPS or LTA specific antibodies followed by protein A Sepharose beads. The receptorLPS-antibody complexes were then analyzed by twodimensional (2D) gel electrophoresis. Here we report the novel interactions of bacterial LPS and also LTA with at least two cell surface molecules other than CD14, a 70 kDa and an 80 kDa LPS/LTA associated proteins (LAP-1 and -2 respectfully). Both proteins are implicated in binding events of LPS on both CD14⫹ and CD14⫺ cells. METHODS AND MATERIALS Materials ReLPS from Salmonella Minessota Re595 was purchased from List Labs (Campbell, CA, USA). Phenol extracts of Streptococcus mutans bacterial cell walls suspensions known as LTA was purchased from Sigma Chemical Company (St. Louis, MO, USA). All fine chemicals and human pooled serum were purchased from Sigma Chemical Company as well. Hybridoma cells secreting 26ic (antiCD14) was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Recombinant human LBP [27] and sCD14 were provided by XOMA (US) LLC (Berkeley, CA, USA). Polyclonal serum against LPS was obtained from Biostride (Redwood City, CA, USA). MAb against LTA of Streptococcus mutants was obtained from Chemicon (Harrow, United Kingdom). Rabbit anti-bovine serum albumin-horseradish peroxidase conjugated was obtained from Research Diagnostics (USA). CD55 specific mAb was obtained from Serotec (Oxford, United Kingdom). Cells Chinese Hamster Ovary (CHO) cells transfected with hCD14 cDNA in the expression vector pRc/RSV (CHOCD14), and the following deletion mutants: CHOCD14D2, CHO-CD14D3, CHO-CD14D4, CHOCD14D5, CHO-CD14D7, CHO-CD14D4.7.5 all transfected with hCD14 deleted respectively for DDED, PQPD, DDED⫹PQPD, DPRQY, AVEVE, and DDED⫹PQPD⫹AVEVE⫹DPRQY amino acids at the N-terminus of CD14 were kindly provided by Dr. S. Viriyakosol and Dr. T. Kirkland (University of California) [28]. CHO cells were maintained in DMEM/F12 (Dulbecco’s Modified Eagle’s Medium/Ham’s F12 1:1 mix) from Gibco BRL (Grand Island, NY, USA) with 2 mM L-glutamine, 7.5% FCS, 500 ␮g/ml gentamycin sulfate (G418; Sigma). Cells were grown in 80 cm3 tissue culture flasks (Nunc). Trypsin/EDTA (0.05% Trypsin/ 0.53 mM EDTA) was used for passaging the cells.

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FIGURE 1 CD14 expression on CHO wildtype and mutant cells. CHO-CD14 (A), -D2 (B), -D3 (C), -D4 (D), -D5 (E), -D7 (F), -D4.7.5 (G), and ECV-304 (H) cells were surface labeled with biotin-NHS ester and lysed in nonionic detergents. CD14 molecules were immunoprecipitated using CD14 specific mAb 26ic and Protein A Sepharose. The immunoprecipitated material was resolved with NEPHGE using a pH gradient of 3.5–10 as the first dimension, and a 10% polyacrylamide gel electrophoresis as the second. The positions of the molecular weight markers are indicated on the right.

The human umbilical vein endothelial cell line designated ECV-304 was obtained from the ECACC. They were maintained in medium 199 supplemented with Glutamax (Gibco BRL) and 10% FCS. Cells were grown in 80 cm3 flasks (Nunc). Cultures of high viability were obtained by trypsinising the cells in 0.25% Trypsin/

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EDTA, followed by seeding the cells at a density of 1 ⫻ 105 cells per 25 cm3 flask. The MonoMac-6 cell line was obtained from the Institute of Immunology, University of Munich (Munich, Germany). MonoMac-6 cells were cultured in 5% CO2 at 37°C in Iscove modified Dulbecco medium (Gibco BRL) containing 10% foetal calf serum. Cell Surface Labeling CHO cells (1.0 ⫻ 107) were washed three times in ice-cold PBS and then incubated for 30 min at 4°C with 40 ␮l/ml membrane-impenetrable Biotin-NHS reagent (Amersham, Buckinhamsire, United Kingdom). Cells were recovered by gentle centrifugation at 400 g and washed twice with ice-cold PBS. Afterwards, the cells were solubilized in buffer containing 50 mM Tris-HCL, pH 8; 1% Nonidet P-40; 6 mM (3-[(-Cholamidopropyl)-

Association of LPS/LTA with LAP Proteins

FIGURE 2 LPS precipitations. CHO-CD14 (A), -D2 (C), -D3 (D), -D4 (E), -D5 (F), -D7 (G), -D4.7.5 (H), and ECV304 (B) cells were surface labeled with biotin-NHS ester and lysed in nonionic detergents. LPS (10 ng) was added in the presence of 5% HPS, and LPS-receptor complexes were immunoprecipitated with the LPS specific polyclonal serum and Protein A sepharose. As a control, lysates were incubated in the absence of LPS, but in the presence of LPS specific polyclonal serum and Protein A sepharose (I). The immunoprecipitated material was resolved with NEPHGE using a pH gradient of 3.5–10 as the first dimension, and a 10% polyacrylamide gel electrophoresis as the second. The positions of the molecular weight markers are indicated on the right.

dimethylammonio]-1-propanesulfonate; CHAPS); 150 mM NaCl; 5 mM EDTA; 50 ␮M phenylmethylsulfonyl fluoride; 0.1 mM iodoacetatmide; 10 nM leupeptin; 10 nM pepstatin; and 10 ␮g/ml trypsin inhibitor (solubilization buffer) for 1 h at 4°C, 22°C, 37°C, or 50°C. The cell lysates were then cleared of debris by a 20-min centrifugation at 10,000 g and stored at 4°C.

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Immunoprecipitation with CD14 Specific Antibodies Detergent-solubilized lysates of CHO cells surface labeled with biotin-NHS, were precleared with rabbit antimouse immunoglobulin (Dako, The Netherlands) and 10% protein A Sepharose slurry (Pharmacia Biotech, Uppsala, Sweden) in solubilzation buffer. CD14 molecules were precipitated with 2 ␮l of 26ic for 1 h at 4°C followed by incubation with 10% protein A Sepharose slurry for 45 min with intermittent mixing. All precipitates were washed five times with solubilization buffer. Immune complexes were dissociated in 25 ␮l of sample buffer (125 mM Tris-HCl, pH6.8; 4% [w/v] SDS; 20% [w/v] glycerol; 1.4 M ␤-mercaptoethanol 0.1% bromophenol blue) and boiled for 5 min. Eluates were analyzed by 2D electrophoresis consisting of nonequilibrium pH gradient electrophoresis (NEPHGE) with a pH gradient of 3.5–10 as the first dimension, and 10% SDS-PAGE as the second dimension.

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FIGURE 3 LTA precipitations. CHO-CD14 (A and B) and ECV-304 (C and D) cells were surface labeled with biotinNHS ester and lysed in nonionic detergents. LTA (10 ng) was added in the presence of 5% HPS (A and C) and LTA receptor complexes were immunoprecipitated with the LTA specific mAb and Protein A sepharose. As a control, lysates were incubated in the absence of LTA, but in the presence of LTA specific mAb and Protein A sepharose (B and D). The immunoprecipitated material was resolved with NEPHGE using a pH gradient of 3.5–10 as the first dimension, and a 10% polyacrylamide gel electrophoresis as the second. The positions of the molecular weight markers are indicated on the right.

Immunoprecipitation with LPS/LTA CHO and ECV-304 cells were cell surface labeled with biotin-NHS reagent and lysed using nonionic detergents (NP-40/CHAPS). Following solubilization, lysates were precleared using 5 ␮l of normal rabbit serum, followed by the addition of 10% Protein A Sepharose slurry in solubilization buffer. After preclearing, 10 or 250 ng/ml of LPS or LTA were used as an affinity surface in order to bind the solubilized receptor(s) in the presence of 5% human pooled serum (HPS). The LPS-receptor complexes or LTA-receptor complexes were then immunoprecipitated by an anti-LPS or anti-LTA antibody and Protein A Sepharose beads. All precipitates were washed five times with solubilization buffer. The immunoprecipitated material was then eluted from the beads by incubating them with NEPHGE sample buffer and subsequently analyzed by high resolution 2D electrophoresis. Nonequilibrium pH gradient electrophoresis with a

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pH range of 3.5–10 was used as the first dimension and 10% polyacrylamide SDS-PAGE was used as the second. Nonequilibrium pH Gradient Electrophoresis The first dimensional gel mixture was prepared by adding 5.5 g urea (ultra pure grade), 1.5 ml of NEPHGE acrylamide stock solution (30% w/v acrylamide, 1.8% w/v N,N⬘-methylenebisacrylamide), 2.0 ml NP-40 stock solution (10% w/v NP-40), 0.5 ml Ampholine carrier ampholytes pH 3.5–10 (LKB or Pharmacia Fine Chemicals) and 2.0 ml Milli-Q water. The mixture was blended in a warm temperature (30°C) until it was homogenous and degassed for 10 min. To achieve polymerization, 15 ␮l and 7 ␮l of ammonium persulfate (APS), 10% w/v and N,N,N⬘,N⬘-tetramethylethylenediamine (TEMED) were added, respectively. The gel solution was quickly dispensed into 2 mm glass cylindrical tubes (12 cm in length) sealed with sealing film (Nescofilm) and overlayed with 20 ␮l of Milli-Q-water. The tube gels were allowed to polymerize for exactly 1 h at room temperature. In parallel, the immunoprecipitation pellets were eluted in 20 ␮l of NEPHGE sample buffer (700 ␮l NET buffer [500 mM Tris-HCl, 1.5 M NaCl, 50 mM EDTA], 400 ␮l 10% NP-40, 100 ␮l of ␤-mercaptoethanol, 100 ␮l ampholytes, 0.2 g sucrose, 1.75 g urea) for 1 h at 50°C. Prior to loading the samples the tube gels were primed with 10 ␮l of NEPHGE sample buffer. Once the antigen was loaded it was overlayed with 20 ␮l of NEPHGE overlay buffer (0.48 g urea, 25 ␮l ampholytes, raised to 1 ml with distilled water). The anodic (0.01 M H3PO4, top chamber) and

Association of LPS/LTA with LAP Proteins

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FIGURE 4 Immunodepletion experiment. CHO-CD14 cells were surface labeled with biotin NHS-ester and lysed in nonionic detergents. LPS (10 ng) was added in the presence of 5% HPS. LPSreceptor complexes were immunoprecipitated with an LPS specific polyclonal serum and Protein A sepharose (A). LTA (10 ng/ml) was subsequently added in the presence of 5% HPS in the same lysate. LTA-receptor complexes were immunoprecipitated with an LTA specific mAb and Protein A sepharose (B). The immunoprecipitated material was resolved with NEPHGE using a pH gradient of 3.5–10 as the first dimension, and a 10% polyacrylamide gel electrophoresis as the second. The positions of the molecular weight markers are indicated on the right.

cathodic (0.02 M NaOH, lower chamber) buffers were extensively degassed. The samples were electrophoresed for 2600 V at 200V constant voltage, reverse polarity. The Bio-Rad Tube Gel System (model 175; Bio-Rad, Hercules, CA, USA) was used for the first dimension. The gels were then extruded by water pressure and incubated for 1 h in 2 ml of SDS PAGE equilibration buffer (125 mM Tris-HCl pH 6.8, 4% [w/v] SDS, 20% [w/v] glycerol, 1.4 M ␤-mercaptoethanol) at RT. The gels were continuously rotated and then stored at ⫺20°C, awaiting SDS-PAGE analysis. Western Blotting LPS/LTA immunoprecipitates or purified BSA were analyzed by SDS-PAGE and transferred onto a nitrocellulose filter (Schleicher-Schuell, Germany) or Immobilon P membranes (Millipore, Bedfore, MA, USA) for 1 h at 250 mA in the presence of transfer buffer (20 mM Tris-acetate, 0.1% SDS, 20% isopropanol, pH 8.3). After transfer, the membrane was blocked for 1 h in blocking solution (5% low fat dried milk dissolved in PBS-T) and washed with PBS-T (two rinses, a 15-min wash, and two 10-min washes). The membrane was then incubated with an appropriate dilution of anti-CD14 specific mAb (MY4) conjugated to HRP, or anti-albumin-HRP for the BSA control experiments, whereas streptavidin-HRP was used for all other experiments. After extensive washing with PBS-T, the antigen was visualised using the ECL procedure (Amersham) according to the manufacturer’s instructions.

Time-Course Experiments CHO cells (1.0 ⫻ 107) were washed three times in PBS and then incubated for 0 min, 10 min, 30 min, and 1 h with 10 ng of LPS in the presence of 5% HPS in serum free medium (Gibco), prior to cell surface labeling as described above. Afterwards the cell lysates were either immunoprecipitated by CD14 specific or LPS/LTA specific antibodies. RESULTS CD14 Expression on CHO Wildtype and Mutant Cells Transfected with CD14 We set out to investigate the possibility that cell surface molecules involved in LPS signal transduction could coprecipitate with CD14. Cells were surface-labeled with biotin-NHS, and solubilized in nonionic detergents as described above in order to retain any weak associations. CD14 was immunoprecipitated using the CD14 specific mAb, 26ic, which is known to recognize an epitope on the nonfunctional domain of CD14. The immunoprecipitated material was then analysed by 2D electrophoresis. No cell surface molecules coprecipitated with CD14 either in the CHO-CD14 cells (Figure 1A) or in the deletion mutants (Figures 1B–1G). Endothelial cells, which do not express CD14, were also used as a negative control (Figure 1H). Only different glycosylation states of CD14 were observed among the mutants. In order to verify that the immunoprecipitated material was indeed CD14, the nitrocellulose membranes were western blot-

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FIGURE 5 BSA control experiment. Purified BSA (A and B) and LPS-immunoprecipitated material (LAP proteins) from CHO-CD14 cells (C) were resolved with NEPHGE using a pH gradient of 3.5–10 as the first dimension, and a 10% polyacrylamide gel electrophoresis as the second dimension. BSA (B) and LPS-immunoprecipitated material (C) were then transferred on nitrocellulose and Western blotted using a BSA specific mAb conjugated to HRP. The positions of the molecular weight markers are indicated on the right.

ted using anti-CD14 mAb (MY4) conjugated to HRP (data not shown). It was shown that indeed the immunoprecipitated material was CD14. LPS Precipitation CHO and endothelial cells were cell surface labeled and LPS-bound complexes were immunoprecipitated using 10 or 250 ng/ml LPS as described above. NEPHGE/SDSPAGE analysis of the immunoprecipitates revealed the presence of three major spots at both LPS concentrations. One LPS associated protein with a molecular weight of 70 kDa and an apparent pI of 4.5–5.0. A second protein spot of 80 kDa with apparent pI of 5.0, that was not so prominent at shorter exposures. These two proteins were designated LAP1 and LAP2 respectively. In addition to the major spots, an 18 kDa protein with apparent pI of 4.5 was detected (Figure 2A). As a control, cell surface labeled lysate was incubated with the same amount of HPS and anti-LPS polyclonal serum without the addition of LPS. The control immunoprecipitation was also analyzed by NEPHGE/SDS-PAGE. The first two protein spots did not appear in the absence of LPS, but the third

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one (18 kDa) appeared in the control, showing that it was a nonspecific spot (NS) (Figure 2I). The same protein spots appeared in immunoprecipitates from ECV-304 cells in the presence of 5% HPS (Figure 2B). To determine whether deletions on the N-terminus of CD14 affected the binding of LPS to the LAP proteins we tested whether these proteins could be immunoprecipitated from the CHO mutants. It was shown that less amounts of the LAP proteins were immunoprecipitated from all the mutants. This was in good agreement with previous studies that showed that N-terminus deletion mutants have reduced signalling capabilities, as measured by the activation of NF-␬B [28]. In mutants CHO-D4, -D5, -D7, and -D4.7.5 LAP2 was not immunoprecipitated (Figures 2E–2H) suggesting that amino acids DDED, PQPD, DPRQY, and AVEVE on the N-terminus of CD14 are important for interactions with LPS and LAP-2. In contrast, from mutants D2 and D3 both LAP proteins were immunoprecipitated (Figures 2C and 2D), even though in less amounts than in CHOCD14 and ECV-304 cells. LTA Precipitations In order to test whether the innate immune system recognises gram-negative and gram-positive bacteria via a common receptor, we attempted to precipitate the two LAP proteins from cell surface labeled CHO-CD14 cells and ECV-304 cells. LTA was used as an affinity ligand to bind the solubilized proteins in the presence of 5% HPS.

Association of LPS/LTA with LAP Proteins

FIGURE 6 Serum dependence of LPS binding to LAP proteins. CHO-CD14 (A and B) and ECV-304 (C and D) cells were surface labeled with biotin NHS-ester and lysed in nonionic detergents. LPS (10 ng) was added either in the presence (A and C) or absence (B and D) of 5% HPS. LPS-receptor complexes were immunoprecipitated with an LPS specific polyclonal serum and Protein A sepharose. The immunoprecipitated material was resolved with NEPHGE using a pH gradient of 3.5–10 as the first dimension, and a 10% polyacrylamide gel electrophoresis as the second. The positions of the molecular weight markers are indicated on the right.

The immunoprecipitated LTA-protein complexes were eluted with NEPHGE sample buffer and analyzed by high-resolution 2D electrophoresis. It was revealed that the two LAP proteins could be immunoprecipitated when using LTA from gram-positive bacteria, both in cells possessing mCD14 (Figure 3A) and from epithelial cells which lack mCD14 (Figure 3C). In the absence of LTA, but in the presence of LTA specific mAb and 5% HPS, the LAP proteins did not appear (Figure 3B and 3D), showing that the immunoprecipitated material is specific and that the antibody does not bind nonspecifically. In order to determine whether the two LAP proteins precipitated with LPS and anti-LPS polyclonal serum were the same proteins that were immunoprecipitated also with LTA and LTA specific mAb, we set out to perform immunodepletion experiments. CHO-CD14 cell surface labeled lysate was used to immunoprecipitate the LAP proteins using LPS and anti-LPS polyclonal serum (Figure 4A). Following the LPS precipitation, the same lysate was used again in order to precipitate the

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LAP proteins using LTA and LTA specific mAb (Figure 4B). It was shown that LPS and LTA are utilizing the same proteins, because the LAP proteins were immunoprecipitated in the first instance using LPS (Figure 4A), therefore the lysate was already depleted thus the LAP proteins couldn’t be precipitated using LTA (Figure 4B). The existence of a 70 or 80 kDa protein as a receptor for LPS has been previously suggested [21–23, 29, 30]. It was later confirmed that the 70 kDa protein that Dziarski [31] isolated was cell bound albumin. It is highly unlikely that either one of the two LAP proteins that we have isolated are cell bound albumin. Bovine serum albumin (BSA) has a molecular weight of 66 kDa and a pI of 6.0, which is significantly more basic than the LAP proteins (Figure 5A). Furthermore, in order to confirm that our 70 kDa protein was not cell bound albumin, we Western blotted the LPS/LTA immunoprecipitates with an anti-albumin antibody conjugated to HRP. It was shown that neither one of our LAP proteins was albumin (Figure 5C). As a positive control, BSA was run on a gel and transferred on nitrocellulose membranes, followed by Western blotting with the antialbumin antibody conjugated to HRP (Figure 5B). More recently El-Samalouti et al. [32] have identified an 80 kDa LPS binding protein (LMP80) as decay accelerating factor (DAF, CD55). In order to investigate whether either one of the LAP proteins that we had isolated were CD55, we immunoblotted LPS immunoprecipitates with CD55 specific mAb followed by incubation with rabbit antimouse Ig conjugated to HRP. Our results showed that neither of the LAP proteins were CD55 (data not shown).

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FIGURE 7 sCD14 dependence of LPS/LTA-LAP interactions. ECV-304 cells were surface labeled with biotin NHSester and lysed in nonionic detergents. In the presence (A and C) or absence (B and D) of 1 ␮g of sCD14, 10 ng of LPS (A and B) or LTA (C and D) was added. LPS/LTA-receptor complexes were immunoprecipitated with either an LPS specific polyclonal serum and Protein A sepharose or an LTA specific mAb and Protein A sepharose. The immunoprecipitated material was resolved with NEPHGE using a pH gradient of 3.5–10 as the first dimension, and a 10% polyacrylamide gel electrophoresis as the second. The positions of the molecular weight markers are indicated on the right.

are required for the interaction of LPS with the LAP proteins in cells lacking mCD14. The serum dependance of the LTA-LAP interaction was also tested. CHO-CD14 and ECV-304 cells were surface labeled and lysed. The cell lysate was immunoprecipitated with 10 ng/ml of LTA in the presence or absence of 5% HPS. The LTA specific mAb was able to immunoprecipitate the LTA-LAP complex only in the presence of serum, in both cell lines, showing that the LTA-LAP interactions are also serum dependent (data not shown).

Serum Dependence of LPS and LTA Binding to LAP Proteins The CD14-LPS interaction is greatly enhanced in the presence of serum or LBP. In order to test whether the LPS/LTA interactions with the LAP proteins are also serum dependent, we attempted to precipitate the LAP proteins either in the presence or absence of serum. CHO-CD14 cells were surface labeled, lysed, and immunoprecipitated with 10 ng/ml of LPS in the presence (Figure 6A) or absence (Figure 6B) of 5% HPS. The LAP proteins failed to be immunoprecipitated in the absence of serum, even though mCD14 was present on the cells (Figure 6B). ECV-304 cells were also used as a control. In the presence of serum both LAP proteinswere immunoprecipitated (Figure 6C), which in the absence of 5% HPS the LAP proteins did not appear (Figure 6D), suggesting that sCD14 and possibly LBP from the serum

LBP Dependence of LPS/LTA-LAP Interactions Although we established that the LPS/LTA-LAP interactions are serum dependent, we set out to test whether this serum dependence is due to LBP or another serum protein. CHO-CD14 cells were surface labeled and lysed in nonionic detergents. The LAP proteins were not immunoprecipitated with 10 ng/ml of LPS either in the presence or absence of 1 ␮g/ml of LBP, suggesting that LBP alone is not sufficient for the LPS-LAP interactions. When 10 ng/ml of LTA were used to immunoprecipitate, the results were identical (data not shown). The two LAP proteins failed to be immunoprecipitated, suggesting that since LBP alone is not sufficient for LPS/LTALAP interactions, another serum protein(s) is possibly required. LBP concentrations ranging from 5 to 100 ng/ml also gave similar results (data not shown).

Association of LPS/LTA with LAP Proteins

FIGURE 8 Mono-mac 6 cells and LPS-LAP interactions. Mono-mac 6 cells were surface labeled with biotin NHS-ester and lysed in nonionic detergents. In the presence (A) or absence (B) of 5% HPS, or with in the presence (C) or absence (D) of 1 ␮g of LBP, 10 ng of LPS was added. The LPS-receptor complexes were immunoprecipitated with an LPS specific polyclonal serum and Protein A sepharose. The immunoprecipitated material was resolved with NEPHGE using a pH gradient of 3.5–10 as the first dimension, and a 10% polyacrylamide gel electrophoresis as the second. The positions of the molecular weight markers are indicated on the right.

Soluble CD14 Dependence of LPS/LTA-LAP Interactions Complexes of soluble CD14 and LPS are known to activate cells that lack mCD14 via a yet unknown receptor. We set out to determine whether the LPS/LTA interactions with the LAP proteins in cells lacking mCD14 are dependent on sCD14. ECV-304, a human umbilical vein endothelial cell line was surface labeled and lysed. We attempted to immunoprecipitate the LAP proteins with 10 ng/ml of LPS either in the presence of 1 ␮g/ml of rsCD14 (Figure 7A) or absence (Figure 7B). The LAP proteins were only immunoprecipitated in the presence of sCD14, suggesting the involvement of CD14 in the LPS-LAP interactions. The same results were obtained with 10 ng/ml LTA in the presence (Figure 7C) or absence (Figure 7D) of 1 ␮g of rsCD14, showing that the LAP proteins appeared only in the presence of sCD14. Mono-mac 6 Cells and LPS/LTA-LAP Interactions Monocytes are of particular importance in the host’s response to LPS during endotoxemia. In order to verify

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our findings, we used Mono-mac 6, a human monocytic cell line, to precipitate the LAP proteins. Mono-mac 6 cells were surface labeled with biotinNHS and lysed in nonionic detergents. The cell lysate was incubated with 10 ng/ml of LPS either in the presence (Figure 8A) or absence (Figure 8B) of 5% HPS, or with 1 ␮g of rLBP (Figure 8C). As a negative control the lysate was incubated without LPS, but with LPS specific polyclonal serum and protein A sepharose (Figure 8D). Only in the presence of 5% HPS were the LAP proteins visible. In the absence of 5% HPS and in the presence of 1 ␮g LBP, only the nonspecific spot appeared. The same results were obtained when using LTA to precipitate the two proteins (data not shown). Coprecipitation of CD14 and the LAP Proteins Although CD14-expressing cells were used for most of our experiments, it was puzzling that we were not precipitating CD14 with the LAP proteins. In an attempt to detect the association, we incubated the CHO-CD14 cells with 10 ng/ml of LPS in the presence of 5% HPS over a range of different time points, prior to cell surface labeling the cells. The cell lysate was subsequently used to precipitate the LAP proteins using and anti-LPS polyclonal serum. It was shown that at 0 min (Figure 9A), 10 min (Figure 9B), and 30 min (Figure 9C) CD14 was coprecipitated with the LAP proteins. After 1 h of preincubation, CD14 and the two LAP proteins could still be immunoprecipitated but two new proteins were also detected (Figure 9D). One protein spot with a molecular weight of 55 kDa and apparent pI of 5. A second protein

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FIGURE 9 Time course experiment. CHO-CD14 cells were incubated with 10 ng of LPS in the presence of 5% HPS for 0 min (A), 10 min (B), 30 min (C), or 1 h (D) prior to surface labeling with biotin NHS-ester and lysis in nonionic detergents. LPS-receptor complexes were immunoprecipitated with an LPS specific polyclonal serum and Protein A sepharose. The immunoprecipitated material was resolved with NEPHGE using a pH gradient of 3.5–10 as the first dimension, and a 10% polyacrylamide gel electrophoresis as the second. The positions of the molecular weight markers are indicated on the right.

spot of 40 kDa with apparent pI of 5.5– 6 was also detected. These two proteins were designated LAP3 and LAP4, respectively. Similar results were obtained when using LTA for the immunoprecipitations (data not shown). DISCUSSION During the past decade numerous researchers have tried to shed light onto the molecular basis behind innate recognition of bacteria. The discovery that CD14 and LBP participate in LPS-induced cell activation stirred a lot of interest, because CD14 is a GPI anchored protein that lacks a transmembrane domain and cannot deliver a signal to the cytoplasm. Thus a model has been suggested where the LPS receptor is a multimeric receptor, consisting of CD14 as the binding domain, and subsequent transmembrane molecule(s) as the signal “transducer” [6]. Much research has been focused in identifying these additional components of the LPS receptors. Many different techniques have been used in this quest. These include photoaffinity crosslinking with

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I-ASD-LPS [21, 23, 33], LPS-coated magnetic particles [24], SDS-PAGE followed by transfer on nitrocellulose and incubation with LPS [25, 26], and finally methods using 3H-labeled LPS [34]. All these methods have the disadvantage that the LPS has been modified (i.e., 125 I-ASD-LPS, 3H-labeled LPS), possibly changing its conformation and affinity to receptors leading to artificial binding [31]. In this study we have chosen to identify possible components of the LPS receptor and investigate whether bacterial LTA binds to the same receptor. Here we describe a biochemical method for the detection of potential LPS/LTA receptor(s) using LPS/LTA concentrations similar to those found in the blood of patients with sepsis [35], in order to closely mimic the physiologic conditions. By using LPS/LTA as an affinity ligand, we have managed to immunoprecipitate two distinct proteins, which we have designated LAP-1 and LAP-2, with molecular weights of 70 kDa and 80 kDa, respectively. To further characterize whether these proteins are part of the LPS multimeric receptor and to test whether different CD14 epitopes are involved in LPS binding, a series of CD14 N-terminus deletion mutants were also used. The immunoprecipitated material (LAP proteins) from the mutants was less than that from the CHOCD14, suggesting that the mutants are able to associate with the two LAP proteins to a lesser extent. This is in good agreement with previous studies that have shown that N-terminal CD14 deletion mutants have reduced signaling capabilities [28]. In mutants D4, D5, D7, and D4.7.5 the LAP-2 protein was not immunoprecipitated, suggesting that amino acids DDED, PQPD, DPRQY,

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TABLE 1 Summary of LPS/LTA immunoprecipitation experiments Cells CHO-CD14 CHO-CD14 CHO-CD14 CHO-CD14 CHO-CD14 CHO-CD14 CHO-D2 CHO-D3 CHO-D4 CHO-D5 CHO-D7 CHO-D4.7.5 ECV-304 ECV-304 ECV-304 ECV-304 ECV-304 ECV-304 Mono Mac 6 Mono Mac 6 Mono Mac 6 Mono Mac 6 Mono Mac 6 Mono Mac 6

Precipitated with LPS LPS LPS LTA LTA LTA LPS LPS LPS LPS LPS LPS LPS LPS LPS LTA LTA LTA LPS LPS LPS LTA LTA LTA

Presence of 5% HPS

Presence of 1 ␮g/ml rLBP

Presence of 1 ␮g/ml rsCD14

Protein precipitated

⻫ — — ⻫ — — ⻫ ⻫ ⻫ ⻫ ⻫ ⻫ ⻫ — — ⻫ — — ⻫ — — — — ⻫

— — ⻫ — — ⻫ — — — — — — — — — — — — — — ⻫ — ⻫ —

— — — — — — — — — — — — — — ⻫ — — ⻫ — — — — — —

LAP1 & LAP2 — — LAP1 & LAP2 — — LAP1 & LAP2 LAP1 & LAP2 LAP1 LAP1 LAP1 LAP1 LAP1 & LAP2 — LAP1 & LAP2 LAP1 & LAP2 — LAP1 & LAP2 — — — — LAP1 & LAP2

All cells were surface labeled with biotin NHS ester and lysed in non-ionic detergents. The immunoprecipitated material was resolved with NEPHGE using a pH gradient of 3.5–10 as the first dimension and 10% SDS-PAGE as the second.

and AVEVE from the N-terminus of CD14 are critical for LPS binding and interaction with a part of the putative receptor, LAP-2. Possibly the conformational change of CD14 after the deletion of these amino acids changes its ability to interact with the putative receptor. Because it has been suggested that CD14 can also bind peptidoglycan from gram-positive bacteria, we decided to test the hypothesis that the innate immune system might utilize the same multimeric receptor in order to recognize both gram-positive and gram-negative bacteria. This hypothesis was confirmed when LTA from gram-positive bacteria was able to precipitate both LAP proteins in the presence of 5% HPS. We further verified that the proteins immunoprecipitated by LPS are the same as the ones precipitated by LTA, by performing immunodepletion experiments. Human pooled serum was found to be essential for the LPS/LTA-LAP interaction. Purified recombinant human LBP was not able to substitute for serum when CHOCD14 and Mono-mac 6 cells were used, both of which express membrane bound CD14. In addition, sCD14, either alone or in combination with rLBP, were sufficient to promote binding in experiments with CHO-CD14. Thus, with mCD14 bearing cells neither sCD14 nor rLBP could replace serum, suggesting that some other serum protein(s) possibly enhances this interaction. Se-

rum was also required for the LPS/LTA-LAP interaction in lysates from ECV-304 cells that lack mCD14. However, in this case it was shown that the LAP proteins could be immunoprecipitated when the serum was replaced by purified recombinant sCD14. The LPS/LTA immunoprecipitation experiments are summarized in Table 1. Due to differences in pI and molecular weight, the two LAP proteins are unlikely to be members of the TLR family that have recently been shown to be involved in LPS signal transduction [8]. Furthermore, the proteins we have identified are not BSA, which has been shown to bind 125I-ASD-labeled LPS and peptidoglycan [31]. BSA has a molecular weight of 66 kDa and a pI of 6.0, which is significantly more basic than the LAP proteins. In addition, we did not detect albumin by Western blotting analysis. Finally Dziarski et al. [31] have demonstrated that the binding of 125I-ASD-labeled-LPS to BSA was not dependent on the presence of serum, whereas the binding of LPS to the LAP proteins is serum dependent. Overall our data suggests that the two LAP proteins that we have isolated are distinct from the toll-like receptors and BSA that have been previously implicated in LPScell interactions. In addition, we have shown that neither one of the LAP proteins is CD55, which has recently been identified as an LPS-binding protein [32].

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The results of this study provide direct evidence that the two LAP proteins represent another unit of the putative CD14-dependent multimeric receptor for LPS and LTA. Binding of LPS or LTA to the LAPs required either functional mCD14, or sCD14 for cells that lack mCD14. These results suggest that LPS (or LTA) first binds to CD14 and is then transferred to the LAPs. When intact CHO-CD14 cells were incubated with LPS and 5% HPS over a range of different time points prior to labeling we were able to coprecipitate CD14 with the LAP proteins using anti-LPS polyclonal serum. After 1 h, LPS bound to two new LAP proteins. Thus, the innate recognition of bacteria might be more complex than previously thought, involving many cell surface molecules. How binding of LPS to the various molecules eventually results in signalling via the TLRs remains unclear. This study represents an important step towards unravelling the host immune response to bacterial infection. Here we demonstrate the existence of a common multimeric receptor (LAP-1 and LAP-2) that requires the presence of either mCD14 or sCD14 for recognition of bacterial components. By gaining a better understanding of the multiple receptors involved in bacterial recognition, in the future clinically useful therapeutic approaches can be developed for the treatment of sepsis and septic shock. ACKNOWLEDGMENTS

This work was supported by the BBSRC. We thank Professor Nelson Fernandez, Dr. Keith M. Wilson, and Professor R. J. Cherry for useful discussions; Drs Suganya Viriyakosol and Theodore Kirkland for kindly providing us with the CHO transfectants. We also thank XOMA (US) LLC for providing us with rsCD14 and rLBP.

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