CD55/decay accelerating factor is part of the lipopolysaccharide-induced receptor complex

2.1 LPS induces translocation of NF-κB in CD55-transfected CHO cells

To evaluate a functional role for CD55 in LPS-induced cell activation, we analyzed the ability of CD55-transfected CHO cells to translocate NF-κB in response to stimulation with LPS (Fig. 1A). Control stimulations were done with CHO cells transfected with the empty expression vector lacking CD55-cDNA. The stimulation of CHO-CD55 cells with LPS resulted in a marked increase of NF-κB translocation, starting at an LPS concentration of 10 ng/ml. This activation of CHO-CD55 cells was observed in all of three established CD55-transfected CHO cell clones as well as in the batch cell line (data not shown). In addition, activation of both cell lines by LPS leads to phosphorylation of IκB-α (Fig. 1C). All CHO-CD55 cells were less sensitive to LPS than CHO-CD14 cells (Fig. 1A, C; 3 and 4D). In contrast to CHO-CD55 and CHO-CD14 cells, control transfectants did not show any NF-κB-response. However, all cell lines showed the same reactivity for murine TNF-α (Fig. 1B).

2.2 Activation of CHO transfectants by LPS and lipid A preparations

Experiments with LPS types from various Gram-negative bacterial strains revealed that CD55-transfected cells respond in general to all LPS preparations and even to purified free lipid A with the translocation of NF-κB (Fig. 2A). To exclude that bacterial membrane compounds, which may contaminate bacterial LPS and free lipid A preparations 23, are responsible for the activation observed, we performed experiments with a synthetic hexa-acylated E. coli type lipid A (compound 506). We found a dose-dependent activation of NF-κB in CHO-CD55 cells by compound 506, starting at a concentration of 50 ng/ml (Fig. 2B).

2.3 Activation of mitogen-activated protein kinases after LPS stimulation

It has been demonstrated that the activation of MAP kinases plays a crucial role in the activation of human monocytes by LPS 24–26, which is also seen in CHO cells expressing CD14 27. Therefore, we investigated the effect of LPS stimulation on the phosphorylation of the three major MAP kinases p38, JNK and ERK in CHO-CD55 in comparison to CHO-CD14 transfectants.

Both cell lines showed increased phosphorylation of p38 in a dose-dependent manner. Consistent with the results from the NF-κB experiments, CHO-CD14 cells appeared to be more sensitive to LPS than CHO-CD55 cells (Fig. 3, top panels). Basically, the same result was obtained when JNK and ERK phosphorylation was analyzed: again, CHO-CD14 cells responded to lower LPS doses than CHO-CD55 cells (Fig. 3, middle and lower panels).

2.4 Effect of LPS antagonists on the activation of CHO-CD55 and CHO-CD14 cells

Since we could only detect quantitative, but not any qualitative differences in the signaling pathways induced by LPS in CHO-CD14 compared to CHO-CD55 cells, it seemed likely that both cell lines signal through TLR4. We wanted to confirm our hypothesis by using TLR4-specific LPS antagonists. The species specificity of LPS antagonists and lipid A partial structures is a well documented feature in human, mouse as well as hamster cells 28. Recently, it has been shown that this species-specific activity depends on the origin of the TLR4 molecule, indicating a direct interaction of these antagonists with TLR4 29, 30. The synthetic tetra-acylated lipid A precursor, compound 406 (also known as lipid IVa), is a well established LPS antagonist in human cells 31–34, but also activates mouse cells, CHO-CD14 cells 28 and CHO-CD55 cells (Fig. 4b). This strongly hints towards TLR4 being the signal transducer in CHO-CD55. The structure of the LPS antagonist B1233 differs from compound 406 (Fig. 4a) by the replacement of the hydroxyl groups at the C-6′-position and the 3′-acyl chain by MeO groups, and the ester linkage of the fatty acyl side chains at the 3- and 3′-positions by ether linkages. The 3- and 3′-acyl groups are shortened to C10, and a double bond has been introduced in the 2′-acyl chain, which has been extended to C18. With the exception of a hydroxy group instead of a 3-keto group at the 2-acyl chain, B1233 is identical to B1287, an LPS antagonist capable of blocking LPS responses in CHO-CD14 and CHO-CD11b cells 35.

Preincubation of CHO-CD14 as well as CHO-CD55 cells with compound B1233 completely blocked the LPS-induced translocation of NF-κB (Fig. 4c). However, it appears that the CD55-transfected cells are more sensitive to the LPS antagonist than the CD14-expressing cells. In order to further strengthen the hypothesis that TLR4 is the signal transducer in CHO-CD55 cells, we transiently transfected CHO-CD14 as well as CHO-CD55 cells with a plasmid encoding for a dominant-negative version of murine MyD88 (DN-MyD88) 36 together with an NF-κB-dependent luciferase reporter construct. In both cell lines, co-expression of DN-MyD88 severely impairs the LPS-induced activation of NF-κB as compared to cells transfected with the empty vector, whereas the TNF-induced NF-κB activation is not significantly changed (Fig. 4D).

2.5 Interaction of free lipid A with CD55 and CD14 as determined by co-immunoprecipitation from CHO-CD55 or CHO-CD14 cells

The results presented above suggest an interaction of lipid A with CD55 in the membrane of CHO-CD55 cells as a prerequisite for activation of the cells. To experimentally prove this hypothesis, co-immunoprecipitation experiments were performed with lipid A and anti-lipid A mAb using CHO-CD55 or CHO-CD14 cells. Cell membranes were treated with lipid A in the presence of human serum, and immunoprecipitation of lipid A-binding proteins was performed with the anti-lipid A mAb A6. Subsequently, the precipitates were analyzed for the presence of CD55 or CD14, respectively. As shown in Fig. 5, CD55 could be co-immunoprecipitated from the cell membranes of CHO-CD55 cells, whereas CD14 was co-immunoprecipitated from the cell membranes of CHO-CD14 cells. In addition to the CD55 or CD14 bands, further slower moving bands are detectable. These bands represent fragments of the anti-lipid A precipitation antibodies, which were also detected by the secondary enzyme-conjugated goat anti-mouse antibody in Western blot. To control the specificity, co-immunoprecipitation was also performed by the use of LPS plus anti-lipid A mAb, or lipid A plus an isotype control mAb. In both controls neither CD55 nor CD14 was found to be co-precipitated with lipid A.

2.6 CD55 is in close proximity to CD14 and CD11/CD18 upon stimulation of human MNC

To give further evidence for a participation of CD55 in the LPS receptor complex in natural LPS-responsive cells, we investigated if CD55 clusters with other LPS-binding receptors upon stimulation with LPS or Lipid A. We used fluorescence resonance energy transfer (FRET) analysis under two different settings. First, in a whole blood assay (Fig. 6A), we demonstrated LPS-specific clustering of CD55 with CD11b, another receptor that has been shown to interact with LPS 37. Secondly, using isolated human mononuclear cells, FRET analysis indicated low, but significant, close proximity of lipid A with CD55 (Fig. 6B). As a positive control, the same experiments were performed with CD14 and showed a much stronger signal. However, close proximity of CD13, a molecule with a similar distribution on monocytes as CD14, and Lipid A was not detectable.

4.1 Reagents and antibodies

Unless otherwise indicated all fine chemicals were purchased from Sigma (Deisenhofen, Germany), Serva (Heidelberg, Germany), Merck (Darmstadt, Germany) or Boehringer Mannheim (Mannheim, Germany). The anti-NF-κB p65 polyclonal antibody and the anti-phospho-JNK mAb were obtained from Santa Cruz Biotech. (Santa Cruz, CA). All other anti-MAP-kinase polyclonal antibodies as well as the phosphor-IκB Ab were from NEB (Schwalbach, Germany). IgG 1 isotype control antibodies were purchased from Sigma. The anti-CD55 mAb BRIC 110 was from Biozol (Eching, Germany). Anti-CD14 mAb, clone MEM-18, was a kind gift from V. Horejsi (Institute of Molecular Genetics, Prague, Czech Republic) 55. 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 56. This mAb was kindly provided by L. Brade (Borstel, Germany). Horseradish peroxidase-conjugated goat anti-mouse as well as FITC-conjugated goat anti-mouse mAb were from Dianova (Hamburg, Germany). Normal human sera were obtained from healthy volunteers. LPS derived from various Gram-negative strains was kindly provided by H. Brade (Borstel, Germany). LPS was prepared by the hot phenol-water procedure, purified by repeated ultracentrifugation, and converted into the sodium salt form by electrodialysis 57. Unless otherwise stated, LPS from S. enterica serovar Friedenau has been used. All LPS preparations were solubilized in pyrogen-free distilled water and showed no TLR2 transmittable activity. Synthetic bisphosphorylated hexa-acyl lipid A (E. coli type), also known as compound 506, and tetra-acylated compound 406, alsoknown as lipid IVa or lipid A precursor Ia, were synthesized as described previously 58, 59. B1233, a synthetic lipid A antagonist, was a kind gift of EISAI Research Institute (Andover, MA). Murine TNF-α was obtained from Sigma (Deisenhofen, Germany).

4.2 Cell culture

CHO-K1 cells (DSM ACC110) from the DSMZ (Braunschweig, Germany), were transfected with either pCEP4, pCEP4-CD14 (a kind gift of D. Golenbock, Worcester, USA), or pCEP4-CD55 as described elsewhere 19, and were cultured at 37°C in a humidified atmosphere containing 5% CO₂. Cells were grown in Ham's F12 medium supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and 400 μg/ml hygromycin.

4.3 Cell stimulation

Before stimulation, CHO cells were seeded into 6-well plates (1×10⁶/well) and allowed to adhere overnight in Ham's F12 supplemented with 2% human serum. Cells were stimulated with various amounts of LPS or lipid A for 1 h in Ham's F12 containing 2% human serum.

4.4 Transient transfection

For transient transfection, CHO-CD14 and CHO-CD55 cells were seeded into 24-well dishes at a density of 50,000 cells/well 1 day prior transfection. The expression plasmid coding for a dominant-negative murine MyD88 36 was a kind gift from Dr. T. K. Means (Boston University School of Medicine, Boston, MA); the plasmid coding for an NF-κB-dependent expression of firefly luciferase, ELAM-Luc, 60 was a kind gift from Dr. C. Kirschning, Technical University of Munich (Munich, Germany). The internal control expression plasmid for constitutive expression of Renilla luciferase (pRL-TK) was from Promega (Mannheim, Germany). The following day, cells were transiently transfected with Polyfect (Qiagen, Hilden, Germany) with either 2 μg of either DN-MyD88 or empty vector DNA/well, together with 500 ng/well of the ELAM-Luc plasmid and 25 ng/well of the pRL-TK plasmid according to the manufacturers' protocol. After 36 h of incubation, cell were stimulated with LPS and murine TNF for an additional 6 h, followed by the analysis of induced luciferase activity with the Dual-luciferase reporter assay system (Promega).

4.5 Preparation of nuclear extracts and electrophoretic mobility shift assay

The preparation of nuclear extracts was performed as described elsewhere 61. The oligonucleotide containing the NF-κB-binding sequence of the murine immunoglobulin κ light chain enhancer 62 was obtained from MWG-Biotech (Ebersberg, Germany). The oligonucleotide (1.25 pmol) was end-labeled in the presence of [γ-³²P]dATP using T4-polynucleotidkinase (Boehringer Mannheim, Mannheim, Germany) for 30 min at 37°C. Unincorporated nucleotides were removed using a Nick-S-column (Pharmacia, Freiburg, Germany). Labeled oligonucleotides (7.5 fmol) were used in the DNA-binding reaction containing 4 μg of crude nuclear extract. The binding reaction also contained 1 μg poly/dI-dC, 1 μg poly/dAdT, 4% Ficoll, 1 mM dithiothreitol, 2 mM MgCl₂, 0.03% NP40, and 60 mM KCl. Reactions were incubated for 20 min at 4°C and separated by electrophoresis in 4% polyacrylamide gels containing 0.5× TBE (45 mM Tris-borate, 1 mM EDTA). Gels were run at 200 V for 1.5 h, sealed, exposed overnight to a phosphoscreen and analyzed with a PhosphorImager (Molecular Dynamics, Krefeld, Germany). For electrophoretic mobility shift assay (EMSA), probes where incubated with 1 μl of the anti-NF-κB p65 antibody immediately after incubation with the labeled oligonucleotide.

4.6 Preparation of cell lysates for Western blot analysis

CHO cells (1×10⁶) were stimulated and washed twice with cold PBS. Lysis was performed in 150 μl of electrophoresis buffer by shaking for 20 min. Lysates were mixed extensively and cleared by centrifugation (20,000×g, 30 min). Each probe (15 μl) was subjected to SDS-PAGE under reducing conditions on a 10% gel, followed by blotting of the proteins onto PVDF membranes using a wet blotting apparatus for 1 h at 2 mA/cm (constant). PVDF membranes were blocked for 2 h at room temperature with TBS containing 5% nonfat dry milk (TBS/M). Membranes were incubated over night with anti-MAP-kinase mAb, anti-phospho-MAP-kinase mAb or anti-phospho-IκB, respectively, in concentrations described in the manufacturers' protocol. Immunoblots were washed four times in TBS/T, incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit mAb (1:2,000) in TBS/M, and developed with the ECL system (Amersham Buchler, Braunschweig, Germany).

4.7 Co-immunoprecipitation of CD55 and CD14

Co-immunoprecipitation of CD55 and CD14 from cell membranes were performed as described previously 19. Cell membranes from CHO-CD55 or CHO-CD14 cells were incubated with 1 μg lipid A and 50 μl serum in 1 ml PBS containing 1 mM PMSF, 2 mM EDTA for 60 min at 4°C on a shaker. The membranes were washed three times (30 min, 36,000×g, 4°C) with PBS containing 1 mM PMSF and 2 mM EDTA, and were solubilized for 30 min at 4°C in 500 μl solubilizing buffer (150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM PMSF, 2 mM EDTA, 1% CHAPS). After centrifugation (30 min, 36,000×g, 4°C), the supernatants were diluted with 500 μl detergent-free solubilization buffer and incubated for 60 min at 4°C with 200 μl of the monoclonal anti-lipid A antibody A6. For precipitation of the antibody-protein complexes, 50 μl of protein A agarose was added and shaken overnight at 4°C. On the next day, beads were washed three times with solubilizing buffer containing0,5% CHAPS. Elution of proteins was achieved by adding 50 μl SDS-PAGE sample buffer to the precipitates and by boiling for 5 min. Immunoprecipitates or a detergent extract of membrane proteins were subjected to SDS-PAGE under nonreducing conditions on a 9% gel and blotted onto nitrocellulose membranes. For detection of CD55 or CD14, anti CD55 mAb, clone BRIC 110 (1:2,000), or anti-CD14 mAb, clone MEM-18 (1:2,000), were used. After subsequent incubation with horseradish peroxidase-conjugated goat-anti-mouse mAb, the ECL system (Amersham Buchler, Braunschweig) was applied.

4.8 FRET analysis

A dual-laser FACSCalibur flow cytometer and CellQuest software (Becton Dickinson, San José, CA) were used for the measurements. Measurements were performed without compensation. Leukocytes (50,000) were acquired in list mode using forward scatter (FSC) as the triggering signal. Energy transfer measurements were either performed between biotinylated CD55 (clone IA10, PharMingen) and CD11b [clone D12, phycoerythrin (R-PE)-conjugate, BD Bioscience, Heidelberg, Germany] in a whole blood assay or between biotinylated lipid A mAb A6 and CD14-PE (clone RMO52, Coulter/Immunotech, Krefeld,Germany), CD13-PE (clone WM-47, DAKO), or CD55-PE (clone IA10) on isolated human mononuclear cells. Streptavidin-Cy5 (Dianova) was used as secondary reagent for biotinylated antibodies. Cells were stimulated in vitro by LPS (4 ng/ml) or 40 ng/ml synthetic lipid A compound 506 for 15 min. The principle of FRET first described by Förster in the late 1940s 63 allows the measurement of distances between surface molecules. The energy transfer parameter (ETp), which is proportional to FRET efficiency (ET), was calculated according to 1, where A is acceptor, D is donor, FL2 is mean fluorescence in channel 2 (488-530 nm) (donor, R-PE), FL3 is mean fluorescence in channel 3 (488-585 nm), FL4 is mean fluorescence in channel 4 (633-670 nm, acceptor, Cy5) (each value was obtained after autofluorescence subtraction):

(1)