©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Domain in Soluble CD14 Essential for Lipopolysaccharide (LPS) Signaling but Not LPS Binding (*)

(Received for publication, March 23, 1995; and in revised form, May 17, 1995)

Todd S.-C. Juan , Eric Hailman (1)(§), Michael J. Kelley , Samuel D. Wright (1)(§), Henri S. Lichenstein (¶)

From the From Amgen, Inc., Thousand Oaks, California 91320 and the Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD14 is a 55-kDa glycoprotein that binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. Monoclonal antibodies of CD14 such as 3C10 and MEM-18 are known to neutralize biological activity of CD14. Recently, it has been demonstrated that MEM-18 recognizes the LPS-binding site of CD14, between amino acids 57 and 64. It has also been shown that 3C10 recognizes a distinct epitope from that of MEM-18, indicating that 3C10 may yet define another functional domain of CD14. In order to identify the epitope for 3C10, we constructed a series of alanine substitution mutants of soluble CD14 (sCD14). BIAcore analyses showed that regions between amino acids 7 and 10 and between amino acids 11 and 14 are required for 3C10 binding. To assess the effect of altering the 3C10 epitope in CD14, we generated a stable cell line expressing a mutant sCD14 containing alanine substitutions in the region between amino acids 7 and 10, sCD14, and purified this protein to homogeneity. sCD14 has impaired ability to mediate LPS-dependent IL-6 up-regulation in U373 cells, integrin activation in neutrophils, and NF-B activation in U373 cells. Purified sCD14 was, however, capable of forming a stable complex with LPS in an LPS binding protein-facilitated and LPS binding protein-independent fashion. The ability of sCD14 to bind LPS was also demonstrated in assays in which excess sCD14 inhibited LPS-mediated tumor necrosis factor- production in whole blood and adhesion of polymorphonuclear leukocytes to fibrinogen. These data strongly suggest that a region recognized by neutralizing monoclonal antibody 3C10 contains a domain required for cellular signaling but not for LPS binding.


INTRODUCTION

CD14 is a 55-kDa glycoprotein that exists as a glycosylphosphatidylinositol-anchored protein on the surface of monocytes and neutrophils (PMN)()and as a soluble protein found in serum(1, 2, 3, 4) . CD14 binds one molecule of lipopolysaccharide (LPS) in a reaction catalyzed by LPS-binding protein (LBP)(5) , an acute phase serum protein(6) . Complexes of LPS with CD14 have been shown to be sufficient to elicit inflammatory responses in leukocytes (5, 7, 8) and endothelial cells(9, 10, 11, 12) . Deletion mutagenesis studies have demonstrated that the N-terminal 152 amino acids of sCD14 are sufficient for mediating inflammatory responses induced by LPS, suggesting that the LPS binding and cell signaling domains of sCD14 are located within the first 152 amino acids(8) .

Several neutralizing mAbs to CD14 have been shown to antagonize cellular responses to LPS in vitro(1, 5, 9, 12, 13, 14, 15) and in vivo(16) . These mAbs may recognize functional domains of CD14 important for its activity, and studies of the epitopes of these mAbs, therefore, are essential in defining these domains. We have demonstrated that mAbs MEM-18 and 3C10 recognize a sCD14 mutant truncated at amino acid 152, indicating that epitopes for these two mAbs are within the first 152 amino acids(8) . We further characterized the epitope of MEM-18 and defined a region between amino acid 57 and 64 that is essential for LPS binding(17) . Deletion of this region not only disrupted binding of this mAb but also binding of LPS.

The epitope for mAb 3C10 defines another functional domain of CD14. This mAb appears to recognize a different region from that of MEM-18 (8) , and binding of the mAb to sCD14 does not affect LPS binding to sCD14(17) , suggesting that this epitope may be involved in a cellular function other than LPS binding. In this report, we identify the epitope of 3C10 by making a series of site-directed alanine substitution mutants in sCD14. We show that the region between amino acids 7 and 14 is required for 3C10 binding. We further characterized this domain by generating a sCD14 mutant with alanine substituted at amino acids 7-10 (sCD14). This mutant was capable of binding LPS but was impaired in its ability to mediate cellular responses to LPS.


MATERIALS AND METHODS

Reagents

Recombinant soluble CD14 (rsCD14) and recombinant LBP (rLBP) were constructed and purified as described(5) . Concentrations of all purified proteins were determined with a Micro BCA protein kit (Pierce) according to manufacturer's specification. Since full-length rsCD14 terminates at position 348 of the mature protein(5) , we herein refer it as sCD14. The anti-CD14 mAb 3C10 was purified by chromatography on Protein G from the conditioned medium (CM) of a cell line from American Type Culture Collection (ATCC TIB 228). Rabbit polyclonal anti-human CD14 antiserum was prepared as described(8) . Rough LPS (Salmonella minnesota R60 or Re595) and smooth LPS (Escherichia coli 0111:B4 or S. minnesota wild-type) were purchased from LIST Biological Laboratories (Campbell, CA). Enzymes for DNA manipulation were purchased from Boehringer Mannheim.

Site-directed Mutagenesis

Nine alanine substitution mutants of sCD14 were used in this study. Table 1summarizes the names and the amino acid residues substituted in each mutant. The Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA) was used as described previously (17) to generate cDNAs encoding alanine substitution mutants of sCD14 cloned in a mammalian expression vector. The primers used for each mutant are as follows: 5`-CGCCAGAACCTTGTGCAGCTGCCGCTGAAGATTTCCGCTGC-3` for sCD14, 5`-GTGAGCTGGACGATGCAGCTGCCGCCTGCGTCTGCAACTTC-3` for sCD14, 5`-CCGCTGCGTCTGCGCAGCTGCCGCACCTCAGCCCGACTGG-3` for sCD14, 5`-GCAACTTCTCCGAAGCAGCTGCCGCCTGGTCCGAAGCCTTC-3` for sCD14, 5`-GAACCTCAGCCCGACGCAGCTGCAGCCTTCCAGTGTGTG-3` for sCD14, 5`-CCGACTGGTCCGAAGCAGCTGCGTGTGTGTCTGCAGTAGAG-3` for sCD14, 5`-CATGCCGGCGGTGCAGCTGCAGCGCCGTTTCTAAAGCGCG-3` for sCD14, 5`-GGTCTCAACCTAGAGGCAGCTGCAGCGCGCGTCGATGCGGAC-3` for sCD14, and 5`-GAGCCGTTTCTAAAGGCAGCTGCTGCGGACGCCGACCCG-3` for sCD14.



Transient Expression of Mutant sCD14 Proteins in COS-7 Cells

To express mutant sCD14 proteins, mammalian expression vectors containing mutant sCD14 cDNAs were introduced into COS-7 (ATCC CRL 1651) cells by electroporation. Conditions for electroporation and generation of serum-free CM from transfected COS-7 cells were as described(8) . Expression of mutant sCD14 was analyzed by Western blot using anti-CD14 polyclonal antibody.

BIAcore Analyses of Interactions between sCD14 Mutants and 3C10 mAb

Recognition of sCD14 mutant proteins by neutralizing monoclonal antibody 3C10 was performed with a BIAcore biosensor instrument. The instrument, CM5 sensor chips, and amine-coupling kit were purchased from Pharmacia Biosensor. Briefly, mAb 3C10 (200 µg/ml in 20 mM sodium acetate, pH 3.4) was immobilized to a CM5 sensor chip by amine coupling according to the manufacturer's specifications. The flow cell immobilized with 3C10 was then incubated in succession with solutions as detailed in the following steps: step 1, COS-7 CM for 2 min, and step 2, HBS buffer (10 mMN-2-hydroxyethylpiperazine-N`-2-ethanesulfonic acid, pH 7.5, 0.15 M NaCl, 3.4 mM EDTA, 0.005% (v/v) surfactant P20 (Pharmacia Biosensor)) for 2 min. For regeneration, 10 mM HCl solution was injected for 2 min. Injection was performed at a rate of 5 µl/min. To quantitate the binding of sCD14 mutants in COS-7 CM to immobilized 3C10, we calculated the relative response unit (RRU). The RRU was obtained by subtracting the response unit (RU) recorded just before injection of CM from the RU recorded after injection of CM and a 2-min wash.

Purification of sCD14

The expression vector containing the cDNA encoding sCD14 was stably transfected into Chinese hamster ovary cells deficient in dihydrofolate reductase as described(5) . A single clone was grown without serum to generate CM containing sCD14. Mutant protein was purified exactly as described(17) , except immunoaffinity chromatography was performed with anti-CD14 polyclonal antibody coupled to Sepharose 4B (Pharmacia Biosensor). Purity of the sample was checked by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining or Coomassie Blue staining. The changed amino acid sequence was verified through N-terminal sequencing.

U373 Bioassays

Growth of U373 cells (ATCC HTB17, Rockville, MD), activation by purified sCD14 preparations, and quantitation of IL-6 were performed exactly as described(8) . Briefly, mixtures of sCD14 or sCD14 and LPS were added to monolayers of U373 cells in serum-free medium and incubated for 24 h. IL-6 in the supernatant was then measured by enzyme-linked immunosorbent assay.

PMN Adhesion Assays

The ability of rLBP and sCD14 or sCD14 to enable PMN adhesion to fibrinogen-coated plates was assessed by previously established protocols(5, 8) . Briefly, PMNs were incubated for 10 min with LPS, rLBP, and sCD14 or sCD14 and washed, and adhesion to fibrinogen-coated surfaces was measured as described(5, 8) . When smooth LPS is used in this protocol, adhesion is completely dependent on addition of sCD14(8) .

The ability of sCD14 or sCD14 at high concentrations to bind LPS and inhibit LPS-mediated PMN adhesion was also assessed. In this experiment, rough LPS (S. minnesota R60, 10 ng/ml) was incubated with rLBP (1 µg/ml) and the indicated concentrations of sCD14 or sCD14 for 30 min at 37 °C before the addition of PMNs. The adhesion of PMNs was measured as described above.

Electrophoretic Mobility Shift Assays

Whole cell extracts from U373 cells were prepared to assess transcription factor NF-B activation. Cells were seeded in 6-well plates at a density of 1 million cells/well 1 day prior to stimulation. For stimulation, purified sCD14, sCD14(17) , or sCD14 was added at a final concentration of 20 ng/ml with or without 20 ng/ml of Re595 LPS for 20 h. Cells were washed twice with 1 phosphate-buffered saline (Life Technologies, Inc.) and scraped in 200 µl of lysis buffer (20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 1 mM EDTA, 0.5 mM dithiothreitol, 1 mM Pefabloc (Boehringer Mannheim), 5 µg/ml leupeptin, 1 mM sodium orthovanadate, and 2 µg/ml aprotinin) supplemented with 1% Triton X-100 (Sigma). Crude extracts were transferred to microfuge tubes, and debris was separated by centrifugation at 14,000 g for 10 min at 4 °C. Extracts were quickly frozen in liquid nitrogen and stored at -80 °C. Protein concentration of the whole cell extracts was determined by Micro BCA assay and ranged between 1.5 and 2 µg/µl.

For examining the NF-B complexes, we performed electrophoretic mobility shift assays. Two oligonucleotides (5`-CATGGAGGGACTTTCCGCTGGGGACTTTCCAGC-3` and 5`-CATGGCTGGAAAGTCCCCAGCGGAAAGTCCCTC-3`) were annealed to generate a double-stranded DNA containing the NF-B binding site of human immunodeficient virus long terminal repeat promoter(18) . This annealed DNA fragment was then filled in with Klenow fragment (Boehringer Mannheim) and [-P]dCTP (Amersham Corp.) and used as probe at a concentration of 50,000 cpm/lane (about 25 fmol). For binding, 4 µl of whole cell extract was incubated with 4 µl of 5 binding buffer (150 mM Tris-HCl, pH 8.0, 40 mM MgCl, 5 mM dithiothreitol, and 10% glycerol), 2.5 µg of (poly(dIdC)):(poly(dIdC)) (Pharmacia Biosensor), radioactively labeled DNA probe, and an adequate amount of lysis buffer so that the final volume was 20 µl/reaction. The reactions were incubated in a 30 °C water bath for 30 min, and complexes were resolved in a native 4.5% polyacrylamide gel using 0.5 TBE (50 mM Tris-HCl, pH 8.0, 45 mM boric acid, and 5 mM EDTA) at 30 mA for 2 h. The gel was then vacuum-dried at 80 °C for 1 h and exposed to Kodak x-ray film for 20 h. In competition experiments, 100 molar excess of unlabeled NF-B probe was preincubated for 10 min before the addition of radioactive probe.

Native PAGE Assays

To directly assess LPS binding of purified sCD14 preparations, sCD14 or sCD14 was incubated at various concentrations (0, 101, 303, and 909 nM) with 3 µg/ml of H-LPS prepared from E. coli K12 strain LCD25 (List Biological Laboratories) in the presence or absence of 16.7 nM rLBP. The reaction was incubated at 37 °C for 30 min and then electrophoresed on native 4-20% polyacrylamide gels. Gels were prepared for fluorography as described previously(5) .

Inhibition of LPS-Induced TNF- Production in Whole Blood

The ability of sCD14 to bind LPS and inhibit TNF- production in whole blood has been described(19) . Briefly, various concentrations of bovine serum albumin (Miles, New Haven, CT), sCD14, or sCD14 diluted in 50 µl of RPMI medium (Life Technologies, Inc.) were added to 250 µl of freshly drawn blood using heparin as an anti-coagulant. Smooth LPS (S. minnesota wild-type) was added to a final concentration of 0.25 ng/ml. The reaction was incubated at 37 °C for 3 h, and supernatants were obtained by centrifugation at 16,000 g for 2 min. TNF- concentrations in the supernatants were assayed using a Quantikine TNF- enzyme-linked immunosorbent assay kit (R & D Systems, Minneapolis, MN) as suggested by the manufacturer.


RESULTS

Alanine Substitution at Amino Acids 7-10 or 11-14 Disrupts Binding of Neutralizing mAb 3C10 to CD14

3C10 is a mAb that recognizes the N-terminal 152 amino acids of CD14(8) . Previous experiments have shown that 3C10 neutralizes the activity of sCD14(1, 5, 9, 13) . To verify that neutralization of sCD14 activity was due to binding of epitopes within the N-terminal 152 amino acids, we demonstrated that 3C10 inhibited IL-6 production in U373 cells mediated by either sCD14 or sCD14 (data not shown).

To map the epitope for mAb 3C10, a series of alanine substitution mutants were generated by site-directed mutagenesis (Table 1). Plasmids containing cDNA sequences encoding different sCD14 mutants were transfected into COS-7 cells, and CM from these cells were examined for the expression of mutant sCD14 proteins by Western blot (Table 1). With the exception of sCD14, all sCD14 mutants were expressed and secreted by COS-7 cells. BIAcore analysis (Fig. 1) was then used to examine the ability of CM containing mutant sCD14 to bind 3C10. CM containing sCD14 or sCD14 were found not to bind 3C10. These data suggest that the region between amino acids 7 and 14 is involved in recognizing 3C10.


Figure 1: BIAcore analysis of 3C10 binding to alanine substitution mutants of sCD14. CM were collected from COS-7 cells transfected with no DNA (MOCK), sCD14, or sCD14 mutants 4 days after electroporation. All CM were analyzed for their ability to bind 3C10 as described under ``Materials and Methods.'' RRUs were recorded from four repeats of one experiment and calculated as means ± standard deviations.



Purification and Characterization of sCD14

Since neutralizing mAb 3C10 recognized amino acids 7-14, we reasoned that this region of CD14 could play an important role in the biological activity of CD14. To help understand the role of this region, we generated a stable Chinese hamster ovary cell line expressing sCD14 and purified mutant protein from the serum-free CM of this cell line. Purified sCD14 migrated with an apparent M of 55,000 when analyzed by reducing SDS-PAGE (data not shown). N-terminal sequencing indicated that the amino acids between 7 and 10 were replaced with alanine residues as expected.

mAb 3C10 Does Not Recognize Purified sCD14

BIAcore realtime analysis was again used to determine whether mAb 3C10 is able to bind purified sCD14. Fig. 2shows that sCD14 recognized immobilized 3C10 and caused an increase of 1800 RU 2 min after the wash (compare RU of the sensorgram before HCl injection at t = 300 to that before injection of sCD14 at t = 0), confirming previous observations(8) . However, purified sCD14 failed to recognize 3C10 and caused only slight RU change (compare RU of the sensorgram after second wash at t = 750 to that before injection of sCD14 at t = 0) similar to that observed when an irrelevant protein such as bovine serum albumin was injected (data not shown), demonstrating that amino acids 7-10 are required for mAb 3C10 binding.


Figure 2: mAb 3C10 does not recognize purified sCD14. Immobilization of mAb 3C10 to a sensor chip has been described(8) . 10 µg/ml sCD14 or sCD14 was used for injection. Injections of solutions at various ``steps'' are marked on the sensorgram. Wash indicates a washing step using HBS buffer as described under ``Materials and Methods.'' The experiments were performed three times, and the results of one experiment are shown.



sCD14Has Reduced Ability to Mediate Cellular Responses to LPS

To assess the consequences of mutating residues between 7 and 10 in sCD14, we used two previously described assays (5, 8, 9) to measure sCD14 bioactivity. We first examined the ability of sCD14 to enable responses of U373 cells to LPS. Addition of as little as 5 ng/ml sCD14 in the presence of LPS enabled strong IL-6 production (Fig. 3A). In contrast, sCD14 was greatly impaired in its ability to enable responses, and required approximately 10-fold more protein in order to give a similar response to that of sCD14 (Fig. 3A).


Figure 3: sCD14 is defective in enabling cellular responses to LPS. A, sCD14 has reduced ability to stimulate IL-6 production by U373 cells. U373 cells were treated with various concentrations of sCD14 or sCD14 in the presence or absence of LPS (20 ng/ml) for 24 h. IL-6 levels were determined as described(8) . Data presented are means ± standard deviations from four readings in an experiment repeated three times. B, sCD14 but not sCD14 mediates responses of PMN to LPS and LBP. Freshly isolated PMN were incubated with ``smooth'' LPS (E. coli 0111:B4, 30 ng/ml), rLBP (1 µg/ml), and the indicated concentrations of sCD14 or sCD14 for 10 min at 37 °C. Cells were washed, and adhesion to fibrinogen-coated wells was measured(5, 25) . Errorbars indicate standard deviations of triplicate determinations.



We also examined whether sCD14 could enable LPS-induced adhesion of PMN to fibrinogen. Fig. 3B shows that 100 ng/ml sCD14 enabled a strong adhesive response of PMN to smooth LPS and rLBP. However, very little response was seen even when 10,000 ng/ml sCD14 was added. These findings confirm that the region between amino acids 7 and 10 is crucial for the biological activity of sCD14.

sCD14Is Impaired in Its Ability to Activate Transcription Factor NF-B in the Presence of LPS

LPS and sCD14-mediated activation of cells has been shown to involve activation of transcription factors such as NF-B (20, 21, 22) . To assess whether the mutation in sCD14 affected downstream signaling, we examined NF-B activation in U373 cells treated with wild type or mutant sCD14. In the absence of LPS or sCD14, U373 cells possess endogenous NF-B, which forms a complex with labeled NF-B probe (Complex1; Fig. 4, lane1). Stimulation with LPS alone or sCD14 alone caused slight enhancement of NF-B complex 1 and slight induction of a new NF-B complex (Complex 2, Fig. 4, lanes2 and 3), but addition of sCD14 and LPS greatly induced both complexes of NF-B (Fig. 4, compare lanes1 and 4). Both complexes 1 and 2 are NF-B-specific since a 100-fold excess of unlabeled NF-B oligonucleotide preincubated with extracts of U373 cells eliminated formation of both complexes (data not shown). Stimulation of U373 cells with sCD14 and LPS caused only 5% of NF-kB activation as quantitated by gel scanning (Fig. 4, lane6). Comparatively, stimulation of U373 cells with a mutant that does not bind LPS (sCD14) failed to activate NF-B complexes even in the presence of LPS (Fig. 4, lane8). These data indicate that a defect in sCD14 is observed at the level at the transcription factor NF-B. Since activation of NF-B is an early event in signal transduction(26) , these data suggest that sCD14 fails to enable signaling.


Figure 4: sCD14 does not activate NF-B. Whole cell extracts of U373 cells with various treatments (control (lane1), LPS (lane2), sCD14 (lane3), sCD14 and LPS (lane4), sCD14 (lane5), sCD14 and LPS (lane6), sCD14 (lane7), and sCD14 and LPS (lane8)) were obtained, and binding of proteins to the labeled NF-B oligonucleotide was performed as described under ``Materials and Methods.'' Complexes of NF-B were resolved on a native 4.5% polyacrylamide gel. After electrophoresis, the gel was dried and exposed to x-ray film for 16 h. Complexes of labeled probe and NF-B are indicated.



sCD14Forms a Stable Complex with LPS

Reduced signaling by sCD14 could be due to a defect in binding LPS. To directly assess whether sCD14 binds LPS normally, we used a native PAGE assay to detect stable complexes between sCD14 or sCD14 and H-LPS. As previously reported(5) , formation of stable complexes between sCD14 and LPS could be observed after 30 min of incubation (Fig. 5A), and addition of rLBP lowered the concentration of sCD14 required for complex formation (compare Fig. 5B, lane2, with Fig. 5A, lane2). This is consistent with the previous observation (5) that rLBP accelerates the transfer of LPS to sCD14. Interestingly, sCD14 was also able to form stable complexes with H-LPS in the absence of rLBP (Fig. 5A, lanes5-7), and this complex formation was also facilitated by rLBP (compare Fig. 5B, lane5, with Fig. 5A, lane5). These data confirm that sCD14 is capable of binding LPS in an LBP-facilitated and in an LBP-independent fashion in vitro and suggest that the reduced biological activity of sCD14 is not due to an inability to bind LPS.


Figure 5: sCD14 forms stable complexes with H-LPS. Various concentrations of sCD14 (lanes2-4) or sCD14 (lanes 5-7) were incubated with 3 µg/ml H-LPS in the absence (A) or presence of 16.7 nM rLBP (B) as described under ``Materials and Methods.'' Lane1 contains LPS in the absence of additional protein. Mixtures were run on 4-20% native polyacrylamide gels and processed for fluorography. Positions of uncomplexed LPS and complexes between LPS and sCD14 or sCD14 are indicated.



Inhibition of LPS-induced Cellular Responses by High Concentrations of sCD14

To further confirm that sCD14 could bind LPS, we utilized two cell-based assays in which high concentrations of sCD14 prevent LPS-mediated activation of cells. In the first assay, sCD14 or sCD14 was tested for ability to inhibit adhesion of PMN to fibrinogen induced by LPS (Fig. 6A). In this experiment, constant concentrations of LPS and rLBP were incubated with increasing amounts (from 1 to 100 µg/ml) of sCD14 or sCD14. Both proteins were capable of neutralizing LPS and inhibiting the adhesion of PMN induced by LPS.


Figure 6: Inhibition of LPS-induced cellular responses by sCD14. A, inhibition of LPS-induced PMN adhesion by sCD14. Rough LPS (S. minnesota R60, 10 ng/ml) was incubated with LBP and various concentrations of sCD14 or sCD14 at 37 °C for 30 min before addition of PMN. The adhesion of PMN to fibrinogen was measured as described under ``Materials and Methods.'' Errorbars indicate standard deviations from three readings. B, inhibition of TNF- production in whole blood by sCD14. 250 µl of whole blood was incubated with various concentrations of bovine serum albumin, sCD14, or sCD14 in the presence of 0.25 ng/ml smooth LPS (S. minnesota wild type) at 37 °C for 3 h, and TNF- production was measured as described under ``Materials and Methods.'' Fraction of TNF Production refers to the ratio of TNF produced in the presence of exogenous protein divided by TNF produced in the absence of added protein. Errorbars are standard deviations from six readings.



We also examined whether sCD14 could inhibit LPS-mediated TNF- production in a whole blood assay, as has been shown for a recombinant sCD14 expressed in Baculoviridae(19) . Addition of increasing amounts of sCD14 or sCD14 caused inhibition of TNF- production in the whole blood assay (Fig. 6B), while addition of bovine serum albumin did not inhibit TNF- production, confirming the previous observation(19) . These data confirm that sCD14 interacts with LPS as well as sCD14.


DISCUSSION

In this report, we mapped the epitope for neutralizing mAb 3C10 to the region between amino acids 7 and 14 of sCD14. Substitution of alanine residues in this region prevented binding of 3C10 to sCD14. These data are consistent with our previous finding (8, 17) that the 3C10 epitope is located within the first 152 amino acids of sCD14 and is distinct from the epitope of MEM-18 at residues 57-64. To help understand how the 3C10 epitope contributes to CD14 function, we purified sCD14 and showed that this protein was severely impaired in its ability to activate cells. Inability of this protein to promote activation of NF-B suggests that sCD14 fails to support LPS-mediated signaling.

The defect in sCD14 signaling is unlikely to result from an inability of this protein to bind LPS properly or to interact with LBP. sCD14 binds LPS normally, as examined by gelshift (Fig. 5A) and two cell-based assays (Fig. 6), and rLBP facilitates transfer of LPS to sCD14 (Fig. 5B). These data confirm our previous observation that 3C10 binds normally to complexes of sCD14 and LPS(17) . We wish to point out that our experiments have measured direct binding of LPS to sCD14, not the binding of LPS-LBP complexes to cell surface CD14 measured in other reports(1, 23, 27) . Direct binding of LPS to sCD14 is saturable with 1-2 LPS molecules bound per CD14(5) , while binding of LPSLBP complexes is not saturable, with up to 1,000 LPSLBP complexes bound to each CD14 at the cell surface(27) . Stoichiometric LPS-sCD14 complexes stimulate cells, but the precise significance of the interaction of multiple LPSLBP complexes with CD14 is not clear at this time. The above differences may explain our previous observation that mAb 3C10 blocked binding of erythrocytes coated with LPSLBP complexes to macrophages. The antibody may have sterically blocked the approach of large LPSLBP complexes to cell surface CD14. They may also explain the recent observation of Viriyakosol and Kirkland (23) that deletion of amino acids 9-12 disrupted the serum-dependent binding of LPS to CD14-bearing cells. This deletion may have caused conformational changes in CD14 that disrupted direct binding of LPS to CD14, binding of LPS-LBP complexes to CD14, or both. The structural basis for binding of multiple LPS-LBP complexes to CD14 is not clear at this time, but it appears unlikely that a short sequence of amino acids could support binding of 10-1,000 LPSLBP complexes.

Since sCD14 binds LPS normally, its defect in signaling is likely to be manifest at the cell membrane. We (9) and others (10, 11, 12) have postulated the existence of a transmembrane protein that interacts with LPS and/or CD14 and transmits signals to the cytoplasm. It is thus possible that residues 7-10 are essential for the interaction of sCD14 with this transmembrane constituent. Alternatively, sCD14 may be defective in delivering LPS to the lipid bilayer of cells. We have recently shown that sCD14 rapidly shuttles LPS into HDL particles (24) and into phospholipid vesicles,()and it is thus possible that residues 7-10 are essential for delivery of bound LPS into the plasma membrane of cells. Experiments are underway to distinguish these two possibilities.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by United States Public Health Grant AI-30556.

To whom correspondence should be addressed. Tel.: 805-447-3064; Fax: 805-499-9452.

The abbreviations used are: PMN, polymorphonuclear leukocyte; CM, conditioned medium; IL, interleukin; LPS, lipopolysaccharide; LBP, LPS-binding protein; NF-B, nuclear factor B; PAGE, polyacrylamide gel electrophoresis; r, recombinant; s, soluble; RRU, relative response unit; mAb, monoclonal antibody; TNF, tumor necrosis factor.

M. M. Wurfel and S. D. Wright, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Viki Jacobsen for technical support.


REFERENCES
  1. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J., and Mathison, J. C.(1990) Science 249, 1431-1433 [Medline] [Order article via Infotrieve]
  2. Simmons, D. L., Tan, S., Tenen, D. G., Nicholson-Weller, A., and Seed, B.(1989) Blood 73, 284-289 [Abstract]
  3. Bazil, V., Baudys, M., Hilgert, I., Stefanova, I., Low, M. G., Zbrozek, J., and Horejsi, V. (1989)Mol. Immunol. 26, 657-662 [CrossRef][Medline] [Order article via Infotrieve]
  4. Bazil, V., Horejsi, V., Baudys, M., Kristofova, H., Strominger, J. L., Kostka, W., and Hilgert, I.(1986)Eur. J. Immunol. 16, 1583-1589 [Medline] [Order article via Infotrieve]
  5. 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)J. Exp. Med. 179, 269-277 [Abstract]
  6. Tobias, P. S., Soldau, K., and Ulevitch, R. J.(1986)J. Exp. Med. 164, 777-793 [Abstract]
  7. Detmers, P. A., Zhou, D., and Powell, D. E.(1994)J. Immunol. 153, 2137-2145 [Abstract/Free Full Text]
  8. Juan, T. S.-C., Kelley, M. J., Johnson, D. A., Busse, L. A., Hailman, E., Wright, S. D., and Lichenstein, H. S.(1995)J. Biol. Chem. 270, 1382-1387 [Abstract/Free Full Text]
  9. Frey, E. A., Miller, D. S., Jahr, T. G., Sundan, A., Bazil, V., Espevik, T., Finlay, B. B., and Wright, S. D.(1992)J. Exp. Med. 176, 1665-1671 [Abstract]
  10. Pugin, J., Schurer-Maly, C. C., Leturcq, D., Moriarty, A., Ulevitch, R. J., and Tobias, P. S. (1993)Proc. Natl. Acad. Sci. U. S. A. 90, 2744-2748 [Abstract]
  11. Haziot, A., Rong, G.-W., Silver, J., and Goyert, S. M.(1993)J. Immunol. 151, 1500-1507 [Abstract/Free Full Text]
  12. Arditi, M., Zhou, J., Dorio, R., Rong, G.-W., Goyert, S. M., and Kim, K. S.(1993) Infect. Immun. 61, 3149-3156 [Abstract]
  13. Wright, S. D., Ramos, R. A., Hermanowski-Vosatka, A., Rockwell, P., and Detmers, P. A. (1991)J. Exp. Med. 173, 1281-1286 [Abstract]
  14. Dentener, M. A., Bazil, V., von Asmuth, E. J., Ceska, M., and Buurman, W. A.(1993) J. Immunol. 150, 2885-2891 [Abstract/Free Full Text]
  15. Grunwald, U., Kruger, C., Westermann, J., Lukowsky, A., Ehlers, M., and Schutt, C. (1992)J. Immunol. Methods 155, 225-232 [CrossRef][Medline] [Order article via Infotrieve]
  16. Leturcq, D. J., Moriaty, A. M., Winn, R. K., Talbott, G., Martin, T. R., and Ulevitch, R. J. (1994) Satellite Meeting of the 3rd Conference of the International Endotoxin Society22 (abstr.)
  17. Juan, T. S.-C., Hailman, E., Kelley, M. J., Busse, L. A., Davy, E., Empig, C. J., Narhi, L. O., Wright, S. D., and Lichenstein, H. S.(1995)J. Biol. Chem. 270, 5219-5224 [Abstract/Free Full Text]
  18. Nabel, G., and Baltimore, D.(1987)Nature 326, 711-713 [CrossRef][Medline] [Order article via Infotrieve]
  19. Haziot, A., Rong, G.-W., Bazil, V., Silver, J., and Goyert, S. M.(1994)J. Immunol. 152, 5868-5876 [Abstract/Free Full Text]
  20. Sen, R., and Baltimore, D.(1986)Cell 47, 921-928 [Medline] [Order article via Infotrieve]
  21. Lee, J. D., Kato, K., Tobias, P. S., Kirkland, T. N., and Ulevitch, R. J.(1992) J. Exp. Med. 175, 1697-1705 [Abstract]
  22. Bagasra, D., Wright, S. D., Seshamma, T., Oakes, J. W., and Pomerantz, R. J.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6285-6289 [Abstract]
  23. Viriyakosol, S., and Kirkland, T. N.(1995)J. Biol. Chem. 270, 361-368 [Abstract/Free Full Text]
  24. Wurfel, M. M., Hailman, E., and Wright, S. D.(1995)J. Exp. Med., 181,1743-1754 [Abstract]
  25. van Kessel, K. P. M., Park, C. T., and Wright, S. D.(1994)J. Immunol. Methods 172, 25-31 [CrossRef][Medline] [Order article via Infotrieve]
  26. Grilli, M., Chiu, J. J.-S., and Lenardo, M. J.(1993)Int. Rev. Cytol. 143, 1-62 [Medline] [Order article via Infotrieve]
  27. Gegner, J. A., Ulevitch, R. J., and Tobias, P. S.(1995)J. Biol. Chem. 270, 5320-5325 [Abstract/Free Full Text]

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